Autophagy is a process for the bulk degradation system of cytosolic compartments by lysosomes/vacuoles. Autophagosome formation involves dynamic membrane rearrangement for which two ubiquitin-like modifications, Apg12-Apg5 conjugation and MAP-LC3 processing, are essential. In yeast, Apg10p is an E2-like enzyme essential for Apg12p-Apg5p conjugation. However, a mammalian counterpart of yeast Apg10p has not been identified. In this paper, we report the isolation and characterization of a cDNA of the APG10 gene. The calculated molecular mass of mouse APG10 gene product is 24.3 kDa, and the region containing the active-site cysteine is conserved. Apg10p coprecipitates with Apg12p, its substrate, and Apg7p, an E1-like enzyme. A mutation of the active-site cysteine within Apg10 predominantly inhibits Apg12p-conjugation. The coexpression of Apg10p with Apg7p facilitates Apg12p-conjugation. Furthermore, the coexpression of Apg10p with Apg7p facilitates MAP-LC3 modification, while MAP-LC3 is not a substrate of Apg10p. Mammalian Apg10p is ubiquitously expressed in all tissues examined. These results indicate that mammalian Apg10p, an authentic E2-like enzyme essential for Apg12p, plays a pivotal role in the cooperative function of two modification systems.