Mutations in the Drg1/RTP/Rit42 gene are commonly identified in hereditary neuropathies of the motor and sensory systems. This gene was also identified as a p53 target gene and a differentiation-related, putative metastatic suppressor gene in human colon and prostate cancer. In this study, we show that the Rit42 protein is a microtubule-associated protein that localizes to the centrosomes and participates in the spindle checkpoint in a p53-dependent manner. When ectopically expressed and exposed to spindle inhibitors, Rit42 inhibited polyploidy in several p53-deficient tumor cell lines and increased the population of cells in mitotic arrest. Blocking endogenous Rit42 expression by small interfering RNA in normal human mammary epithelial cells resulted in the disappearance of astral microtubules, and dividing spindle fiber formation was rarely detected. Moreover, these cells underwent microtubule inhibitor-induced reduplication, leading to a polyploidy state. Our findings imply that Rit42 plays a role in the regulation of microtubule dynamics and the maintenance of euploidy.