An Assist Trophy is an item that, when grabbed, summons a Nintendo character (or an Unknown Character) out of twenty seven choices of various origin to quickly aid the holder, though there are currently three third party Assist Trophies, not to mention some assistants get in the way more than they help. Assist Trophies work nearly identical to Pokéballs. However, no Pokémon come out of them, and once an Assist Trophy is grabbed, it is automatically activated (though this is not very convenient), assuming the holder is standing on the ground, and not in a jump, attack, or hitstun.
Only one Assist Trophy (and the character it releases) can be in a match at once. The next Assist Trophy will not appear until the data for the character it will release is fully loaded.