Neuroblastoma is a common childhood tumor. Vincristine (VCR), an alkaloid extracted from Catharanthus roseus, is commonly used in combination chemotherapy. However, the mechanisms of VCR-induced neuroblastoma cell death are not clear. The aim of this study was to investigate the impact of VCR on mitosis and apoptosis of SH-SY5Y neuroblastoma cells and the underlying mechanisms. SH-SY5Y cells were treated with increasing VCR doses for different time points. Cell proliferation was detected using the MTT assay. Mitotic rate was quantified by immunofluorescence. Cell cycle and apoptosis were analyzed by flow cytometry. The mRNA and protein expression of caspase-3 and -9 (apoptotic factors), as well as cyclin B and D (cell cycle factors), was evaluated by real-time (RT)-PCR and western blot analysis, respectively. VCR inhibited SH-SY5Y cell proliferation in a time- and dose-dependent manner (P<0.05). The IC50 of VCR in SH-SY5Y cells was determined as 0.1 µM. VCR at 0.1 µM induced mitotic arrest and apoptosis, promoted the expression of caspase-3 and -9 and cyclin B, while decreasing the expression of cyclin D at 6, 12, 18 and 24 h. Except for the mRNA expression of cyclin D at 6 h, these changes were significant at both the mRNA and protein levels (P<0.05). VCR induces mitotic arrest of SH-SY5Y cells by regulating cyclin B and D. It further induces apoptosis in these cells through the activation of caspase-3 and -9. This study provides fundamental evidence for the application of VCR in neuroblastoma chemotherapy.