Texas-Red-asialoorosomucoid (ASOR) fluorescence-sorted early and late endocytic vesicles from rat liver were subjected to proteomic analysis with the aim of identifying functionally important proteins. Several Rab GTPases, including Rab1a, were found. The present study immunolocalized Rab1a to early and late endocytic vesicles and examined its potential role in endocytosis. Huh7 cells with stable knockdown of Rab1a exhibited reduced endocytic processing of ASOR. This correlated with the finding that Rab1a antibody reduced microtubule-based motility of rat-liver-derived early but not late endocytic vesicles in vitro. The inhibitory effect of Rab1a antibody was observed to be specifically towards minus-end-directed motility. Total and minus-end-directed motility was also reduced in early endocytic vesicles prepared from Rab1a-knockdown cells. These results corresponded with virtual absence of the minus-end-directed kinesin Kifc1 from early endocytic vesicles in Rab1a knockdown cells and imply that Rab1a regulates minus-end-directed motility largely by recruiting Kifc1 to early endocytic vesicles.