MUC16 (CA125) is a transmembrane mucin that contributes to the progression of epithelial ovarian cancer (EOC). Expression of MUC16 is not detectable in the epithelial surface of normal ovaries. MUC16 expression is, however, common in serous EOC as well as in metastatic and recurrent tumors. Despite these observations, its contribution to the development of EOC is unknown. We stably expressed either empty vector, MUC16 C-terminal domain (MUC16 CTD) or MUC16 TMU (a construct that lacks the cytoplasmic tail) in NIH3T3 mouse fibroblast cells. In this study, we provide evidence for the role of MUC16 CTD in oncogenic transformation. We show that ectopic expression of MUC16 CTD enhances the growth of NIH3T3 cells under normal and low serum conditions, and promotes anchorage-dependent colony formation. The deletion of the cytoplasmic tail abrogated these effects. MUC16 CTD expression in NIH3T3 cells also enhances the formation of colony in soft agar as compared to MUC16 TMU. MUC16 CTD expression enhances tumor formation in nude mice. Our findings provide the first evidence that MUC16 induces the transformation of NIH3T3 cells and indicate that MUC16 functions as an oncogene. Furthermore, our data suggest that the cytoplasmic tail is critical for MUC16 oncogenic properties.