MOB (Mps one binder) was originally identified in yeast as a regulator of mitotic exit and cytokinesis, and was later identified as a tumor suppressor and a component of an emerging Hippo-LATS tumor suppressor pathway in Drosophila (D). So far, 7 human homologs of yeast MOB (hMOB1A, 1B, 2A, 2B, 2C, 3, 4) have been identified. Although hMOB1A/B has been extensively studied, the biological features of other hMOBs are largely unknown. In addition, while hMOB1 has been reported to interact with and activate LATS (Large tumor suppressor)/Warts tumor suppressor, the functional significance of this is unknown. In this study, we have characterized, for the first time, the cellular and biochemical function of all human MOBs. By examining hMOB mRNAs expression in various human tissues, we found that hMOBs demonstrated different expression patterns. Further biochemical characterization of hMOBs showed that only hMOB1A and hMOB1B interact with both LATS1 and LATS2 in vitro and in vivo. Significantly, we have discovered that overexpression of hMOB1 in human cancer cells activated LATS activity and inhibited cell proliferation or caused apoptosis while hMOB1, targeting the plasma membrane, led to a more significant phenotype. Reciprocally, short-hairpin (sh) RNA-mediated suppression of hMOB1 causes increased cell proliferation. Our findings provided evidence that hMOB1A and hMOB1B are 2 LATS-binding proteins that may function as tumor suppressors in human cancer cells.