Matrine is an active component of Leguminosae plants and is thought to exhibit anti-tumor effects. However, the effects of matrine on drug-resistant cancer have not been fully elucidated. The present study aimed to investigate the effects of matrine on vincristine (VCR)-resistant retinoblastoma (RB) cells and to assess the underlying mechanisms governing this effect. The drug-resistant cell line SO-Rb50/VCR was established by incubation with VCR at increasing concentrations. The effects of matrine on SO-Rb50 and SO-RB50/VCR cell growth and proliferation were evaluated using light microscopy and Cell-Counting Kit-8 assay. In addition, the effects of matrine on cell apoptosis, proliferation and cell cycle staging together with its potential underlying mechanisms were investigated. Matrine inhibited the proliferation of SO-Rb50 and SO-RB50/VCR cells in a concentration-dependent manner (0.2-1.1 mg/ml). However, matrine at the half-maximal inhibitory concentration (IC50) appeared to trigger apoptosis of these cells and had a tendency to arrest the cell cycle at the G0/G1 phase. Matrine treatment also promoted the expression of Bax and reduced the expression of Bcl-2 and cyclin D1 compared with the control. However, matrine was not able to increase the sensitivity of cells to VCR. The results of the present study suggested that matrine has the potential to promote the apoptosis of SO-Rb50/VCR cells and arrest cell cycling, indicating a possible benefit of matrine for the treatment of drug-resistant RB.