Bladder cancer has been considered as one of the most common urinary malignancies. Growing evidence has indicated that Centromere Protein F (CENPF) is a promising molecular biomarker for many human malignant tumors. However, the role of CENPF in bladder cancer (BC) proliferation, migration, invasion, cell cycle and apoptosis thereof remain unclear. In the present study, high-throughput sequencing technology and bioinformatics analysis were conducted to identify mRNAs profiles in 10 pairs of bladder cancer tissues and adjacent noncancerous tissues. CENPF was overexpression in bladder cancer tissues, and higher in MIBC than NMIBC. We investigated the expression of CENPF in 30 other pairs bladder cancers tissues, and the results were in accordance with the sequencing results. Furthermore, Immunohistochemical staining, showed that strong intensity of CENPF in BC tissues than normal tissues. Increased staining of CENPF was detected of tumor cells in MIBC compared with NMIBC. This suggests that CENPF might be highly expressed in aggressive and invasive tumor cells. Subsequently, in vitro functional experiments also demonstrated that the siRNA interference of CENPF expression significantly weakened the proliferation, migration, invasion and apoptosis of BC cells, and the cells were arrested in the G2/S phase in Cell cycle. Moreover, functional enrichment analyses, lncRNA-miRNA-mRNA, and protein-protein interaction networks revealed that CENPF was potentially involved in carcinogenesis and evolution of bladder cancer. Taken together, these results demonstrated that CENPF may serve as a potential biomarker of tumor occurrence, progression, and even prognosis for bladder cancer. However, further research is needed to be further clarified the pathway mechanisms of CENPF in bladder cancer.