Vanadocene dichloride (VDC) was shown to exhibit antitumor properties against a wide spectrum of tumor cell lines. Many studies have been carried out to reveal the bioactivities of VDC and the interaction mechanism of VDC with biological molecules in test tubes. One of the bioactivities of VDC is to arrest the cell cycle at the G2/M phase. However, its underlying mechanisms of action and cytotoxicity profile are still not fully understood. HeLa cells were used in this study, and the IC50 value of VDC was 8.61 μM after a 24-hour treatment. We used an immunofluorescence staining method to analyze the morphology of cells in the mitosis stage to elucidate what defects caused cell arrest in mitosis. Chromosomal misalignment was found to be the major phenotype. One of the proteins responsible for chromosome alignment at the metaphase is Aurora B kinase. Results of immunoblotting assay showed that Aurora B kinase activity was inhibited by VDC treatment. More than 50% of the Aurora B activity was inhibited when cells were treated with VDC at a concentration of 6.25 μM. That VDC was able to induce defects in chromosomal alignment at the metaphase by inhibiting the activity of Aurora B kinase is an important mechanism of VDC to be developed as an antitumor agent.