Activating B-Raf mutations arise in 60% to 70% of human melanomas and are thought to play a vital role in tumorigenesis, although how this occurs remains poorly understood. Wild-type B-Raf is critical for normal mitosis of human somatic cells, suggesting that mutational activation of B-Raf might compromise mitosis. We examined this hypothesis by introducing oncogenic mutant B-Raf(V600E) into established human melanoma cells, assessing the effects on mitosis and their possible relationship to extracellular signal-regulated kinase (ERK) pathway activation. Exogenous expression of this activated B-Raf mutant led to a high incidence of aberrant spindles and supernumerary centrosomes. These mitotic abnormalities were suppressed by expression of a B-Raf(V600E) mutant-specific shRNA or by the addition of the mitogen-activated protein/ERK kinase-specific inhibitor U0126. Mitotic abnormalities generated by B-Raf(V600E) also caused missegregation of chromosomes leading to aneuploidy. Because activating B-Raf mutations are detected frequently in benign nevi, we extended our studies to primary human melanocytes. Remarkably, short-term expression of B-Raf(V600E) was sufficient to induce aneuploidy in human melanocytes or in immortalized human mammary epithelial cells. Collectively, our studies identify a novel role for the B-Raf oncogene in driving aneuploidy in melanocytic cells. We propose that disruption of mitotic controls by oncogenic B-Raf has important implications for understanding melanoma tumor development.