A series of novel indole analogues were discovered as colchicine-binding site inhibitors of tubulin. Among them, 3a exhibited the highest antiproliferative activity (average IC50 = 4.5 nM), better than colchicine (IC50 = 65.3 nM). The crystal structure of 3a in complex with tubulin was solved by X-ray crystallography, which explained the improved binding affinity of 3a to tubulin and thus its higher anticancer activity (IC50 = 4.5 nM) than the lead compound 12b (IC50 = 32.5 nM). In vivo, 3a (5 mg/kg) displayed significant antitumor efficacy against B16-F10 melanoma with a TGI of 62.96% and enhanced the antitumor efficacy of a small-molecule PD-1/PD-L1 inhibitor NP19 (TGI = 77.85%). Moreover, 3a potentiated the antitumor immunity of NP19 by activating the tumor immune microenvironment, as demonstrated by the increased tumor-infiltrating lymphocytes (TIL). Collectively, this work shows a successful example of crystal structure-guided discovery of a novel tubulin inhibitor 3a as a potential anticancer and immune-potentiating agent.