Using in vitro assays with purified proteins, we show that XKCM1 and XKIF2, two distinct members of the internal catalytic domain (Kin I) kinesin subfamily, catalytically destabilize microtubules using a novel mechanism. Both XKCM1 and XKIF2 influence microtubule stability by targeting directly to microtubule ends where they induce a destabilizing conformational change. ATP hydrolysis recycles XKCM1/XKIF2 for multiple rounds of action by dissociating a XKCM1/ XKIF2-tubulin dimer complex released upon microtubule depolymerization. These results establish Kin I kinesins as microtubule-destabilizing enzymes, distinguish them mechanistically from kinesin superfamily members that use ATP hydrolysis to translocate along microtubules, and have important implications for the regulation of microtubule dynamics and for the intracellular functions and evolution of the kinesin superfamily.