Norcantharidin (NCTD), a demethylated form of cantharidin, is currently used as an anti-cancer drug in China. However, the exact anti-cancer mechanism of NCTD on human cancer cells remains poorly understood. In the present study, NCTD inhibited proliferation and DNA replication effectively in HL-60 cells. DNA replication-initiation protein Cdc6 was cleaved after 12 h treatment with NCTD. This cleavage generated a truncated Cdc6 fragment with a relative molecular weight of 49 kDa and elongated treatment with NCTD resulted in a complete loss of Cdc6. In addition, we found that Cdc6 was present in both non-chromatin- and chromatin-bound fractions in the untreated HL-60 cells, and NCTD treatment led to the cleavage of Cdc6 in both fractions. NCTD-induced cleavage of Cdc6 was prevented by pre-treatment with caspase-3 inhibitor, suggesting the involvement of caspase-3 activity in the process. Furthermore, NCTD treatment resulted in apoptotic changes including granular nuclear morphology, DNA laddering and sub-G1 arrest in HL-60 cells. In conclusion, our study reveals that NCTD can inhibit DNA replication, and induce apoptosis and caspase-3-dependent cleavage of Cdc6. The anti-cancer effect of NCTD may be closely associated with the dysfunction of Cdc6 and our report is the first to put forward this point of view.