Objective To investigate the inhibitory effect and mechanism of a novel guanidine derivative, 1-n-pentyl-5-(4-trifluoromethoxyphenyl) biguanide (5C) and osimertinib alone or in combination on bladder cancer.Methods MTT assay and cell colony formation assay were used to detect the effects of 5C and osimertinib alone or in combination on the proliferation and colony formation of bladder cancer cells. Western blotting was used to detect the effects of 5C combined with osimertinib on the epidermal growth factor receptor (EGFR) and its downstream signaling pathways.Results 5C or osimertinib alone significantly inhibited the proliferation and colony formation of bladder cancer cells, and the combination of 5C and osimertinib could better inhibit the proliferation and colony formation of bladder cancer cells. Mechanism studies showed that 5C combined with osimertinib could significantly inhibit the phosphorylation of EGFR and its downstream signaling pathway Akt/Erk.Conclusion 5C combined with osimertinib could inhibit the growth of bladder cancer cells by down-regulating EGFR and its downstream signaling pathway.