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5000 years ago, when the Mushroom Kingdom was young, the Star Spirits were spreading stars across the night sky, making the viewers very happy. But there was one who didn't like the bright shiny stars in the sky. This was Ztarlow, the last of the Ztar Spirits. One night, the Ztar Spirit dashed across the sky, and made the stars disappear, making the viewers sad. The Star Spirits did not like what she had done and imprisoned her in a crystal comet, which crashed into earth. Before she was imprisoned, Ztarlow stated she would return, and that whoever frees her would be her master.
Now in the present, Bowser, Bowser Jr., and the Koopalings were lying on the ground, defeated from they're last encounter with Mario and Luigi. Bowser disliked that they always seem to win. Iggy Koopa suddenly noticed the comet and Bowser broke it open, releasing Ztarlow. Ztarlow tells them that she was prisoned by the Star Spirits, and that now Bowser is her master. Bowser, upon hearing this, laughs evily.
Later at Peach's Castle, Mario, Luigi, Princess Peach, Princess Daisy, and Starlow were having cookies at tea while chatting, when suddenly a rumbling is heard. The five head out to see that Bowser and his sons and daughter were in an airship. With them was Ztarlow, which made Starlow angry. Kamek then appeared and used his Magikoopa magic to put Peach and Daisy in rainbow bubbles made by his magic, and they flew off with the princesses. Mario, Luigi, and Starlow prepared themselves for yet another adventure.
Later in the game, Toadsworth tells them about seven black diamonds that can be helpful.
After defeating Ultimate Ztarlow, she shrieks and self-destructs, scattering pieces of Bowser's Castle. Luckily, Mario, Luigi, Peach, and Daisy managed to get away. After returning to the castle, the Toads and other natives of the Mushroom Kingdom greeted them. Tod even was there, saying how awesome they were. Soon after, the sky turned to night for a peculiar reason, and yellow lights began to glow. They were Star Spirits, all relatives of Starlow. The oldest one said that Starlow must come back. But before Starlow could get too far away, she asked if she could stay in the Mushroom Kingdom with her friends. The oldest one smiled and said she could, making Starlow very happy. And they all lived happily ever after.
Meanwhile, on an island, Wario and Waluigi have landed head-first into the sand due to Ztarlow's self-destruction. Wario got up and dusted himself off. Waluigi got up as well. Then, they're bellies grumbled. They found a crab and decided to cook it. But when they did, the crab's mother, who is extremley huge and muscular, appeared. The screen then went black as punching sounds were heard.
- Princess Peach
- Princess Daisy
- Small Fry Guy
- Unnamed fish resembling Cheep-Cheep
- Unnamed cat-like species
- Tod Toadsty
- Friendly Koopa Troopas
- Unnamed robots resembling Samus
- Shady Bro.
- Friendly Monty Moles
- Larry Koopa - Bowser's second oldest son
- Giant Koopa Troopa - A simply koopa troopa transformed in a giant version by Kamek
- Megaleg - A snifit-like Robot
- Roy Koopa - Bowser's third oldest son
- Goomboss - The king of Goomba
- Vixoomba - A goomba with fox ears and tail
- Lemmy Koopa - Bowser's crazy son. He like Iggy and like apresetation in circus
- Angry Sun - The Super Mario Bros. 3 enemy appeared as boss in this game
- Parapin - A umbrella-like boss
- Wendy O. Koopa - Bowser's only daughter. She's tall with pink shell, she's uses bow, high heels
- Big Bad Bones - A giant dry bones who like the Bad Wolf
- Boolossus - The giant boo from Luigi's Mansion is returned to this game
- Iggy Koopa - Bowser's second craziest son
- Wiggler - A angry-caterpillar
- Jungle Blooper - A green-blooper that live in Jungle's lake
- Morton Koopa Jr. - Bowser's youngest and strongest son. He's dispuct with Roy Koopa
- Fiery Parapin - Parapin is transformed in a Fiery Version
- Lava Piranha - He's returned to this game
- Ludwig Von Koopa - Bowser's oldest son. He composes koopa music
- Big Sky Guy - A little Sky Guy transformed in a giant version by Kamek
- King Lakitu - Giga Lakitu from SMG2, returned as King Lakitu in this game
- Bowser Jr. - Bowser's eighth son. He uses the Magic Paintbrush, transforming into Shadow Mario
- Shadow Mario - Is Mario doppelganger, a negative clone. He uses a hat with letter "M" with the diaeresis in the top.
- Kamek - This Magikoopa transform any thing in other thing
- Bowser - The king of the koopas and the eternal enemy of mario
- Ultimate Ztarlow - Ztarlow reveals the true appearance and she's the final boss of the game
- At one point in the game, Morton calls Peach "Toadstool". Toadstool is really Peach's counterpart from the first three Super Mario Bros. games for the NES.
- This is the first Mario and Luigi game for the Wii.