[Abstract] Objective: To investigate the expression of miR144-3p in bladder cancer tissues and cells and its effect on the proliferation and invasion of T24 cells. Methods: A total of 36 cases of bladder cancer tissue specimens and 10 cases of normal bladder epithelial tissue specimens were collected from Tangdu Hospital of Air Force Medical University during February 2018 and December 2018. In addition, bladder cancer T24 cell line and normal urothelial cell line SV-HUC-1 were also collected for this study. The levels of miR144-3p in bladder cancer tissues and cells were detected by qPCR methods. The miR-144-3p mimics and miR-NC were transfected into T24 cells by LipofectamineTM 2000, respectively. The proliferation, cell cycle distribution and invasion abilities were detected by MTT, Flow cytometry and Transwell chamber methods, respectively. TargetScan software was used to predict the binding site between miR-144-3p and E2F3 (E2F transcription factor 3); Dual luciferase reporter gene assay was used to verify the relationship between miR-144-3p and E2F3; and WB was used to detect the expression levels of miR-144-3p and E2F3 in cells. Results: The expression of miR-144-3p was downregulated in bladder cancer tissues and cells (all P<0.01). In addition, the expression level of miR-144-3p in muscular invasive bladder cancer tissues was significantly lower than that in non-muscular invasive bladder cancer tissues (P<0.05). Dual luciferase reporter gene assay confirmed that there was a targeted relationship between miR-144-3p and E2F3. Overexpression of miR-144-3p inhibited the proliferation and invasion of T24 cells (all P<0.01) and downregulated the expression of E2F3 (P<0.01); upregulation of E2F3 could reverse the inhibitory effect of miR-144-3p overexpression on proliferation and invasion of T24 cells. Conclusion: miR-144-3p has low expression level in bladder cancer tissues. It inhibits proliferation and invasion of bladder cancer cells by downregulating E2F3.