Background: Bladder cancer is one of the most common malignancy in urinary system. Fibroblast growth factor receptor 3 (FGFR3) is the most common alteration gene in bladder cancer, occurring in approximately 70% of non-metastatic bladder cancer and 20% of metastatic bladder cancer. Anlotinib is a potent oral, multi-target tyrosine kinase inhibitor with a favorable safety which mainly targets vascular endothelial growth factor receptor (VEGFR), FGFR, platelet-derived growth factor receptors, and c-kit. This study aims to investigate the role of anlotinib in bladder cancer compared with FGFR3 inhibitor erdafitinib in vitro and in vivo. Methods: MTT, colony formation, and Transwell assays were performed on bladder cancer cells (SW780 and UMUC14) to confirm the effects of anlotinib and erdafitinib on proliferation, migration and invasion. Apoptotic effect was evaluated by Annexin V/propidium iodide double staining, and the levels of the protein and mRNA were examined by RNA-seq, Western blotting and RT-qPCR. Finally, mice with palpable xenografts were treated either with anlotinib and erdafitinib for 8 days before they were sacrificed for measuring the sizes and weights of the tumors. Results: To assess the roles of anlotinib in bladder cancer, we treated SW780 cell line which had FGFR3-BALAP2L1 fusion mutation and UMUC-14 cell line which had a FGFR3 (MuS247C) mutation with DMSO, anlotinib and erdafitinib. As seen in MTT, colony formation and Transwell assays, anlotinib repressed cell proliferation, migration and invasion as erdafitinib. Compared with vehicle, Anlotinib promoted cell apoptosis of bladder cancer cells. To further explore the mechanism, anlotinib and erdafitinib suppressed p-Erk1/2 and p-AKT, while only anlotinib inhibited the expression of VEGF-a. Compared with erdafitinib, the inhibitory ability of anlotinib was weaker than that of erdafitinib in cell line with FGFR3 (MuS247C) mutation, on the contrary, it was much stronger in cell line with FGFR3-BALAP2L1 fusion mutation. In addition, the in vivo data from xenagrafts also supported that anlotinib could significantly repress tumor growth with FGFR3 fusion mutation, and the efficacy was better than erdafitinib. Conclusions: Anlotinib could repress the proliferation, migration and invasion of bladder cancer cells by inhibiting the phosphorylation of Erk1/2 and AKT, and the suppression VEGF-a expression, whose effects was better than erdafitinib in bladder cancer with FGFR3 fusion mutation. Therefore, anlotinib might be a potential novel targeted agent to treat the bladder cancer patients with FGFR3 fusion mutations, and clinical trials are needed for further investigation.