Objective: To establish a melanoma cell line resistant to dacarbazine (DTIC) and to investigate its drug resistance. Methods: O⁶-methylguananine-DNA methyltransferase (MGMT) protein expression of a human melanoma cell line was detected using Western blot. The drug resistance of human melanoma A375 cells to DTIC was induced by in vitro stepwise induction. Drug-resistant cell line IC50 and resistance index were detected by MTT assay. Results: After treated with DTIC at different concentrations, the growth of A375 cells slowed down in different degrees. With the increase of DTIC concentration, the inhibition rate of A375 cells increased in different degrees. The median inhibitory concentration (IC50) of DTIC to in vitro proliferation of parent A375 cells and A375/DTIC cells was 10.766 and 95.693, respectively. The resistance index of A375/DTIC cells to DTIC was 9 times higher than that of A375 cells (P<0.05). Conclusion: The successful establishment of a DTIC-induced drug-resistant human melanoma cell line A375/DTIC in vitro by gradually increasing the concentration of chemotherapeutic drugs with stages lays an experimental foundation for further study on the mechanism of melanoma resistant to DTIC.