Objective: To identify the expression of circNDUFA10 (has_circ_0001118) in bladder cancer (BCa), and explore the underlying mechanisms in inhibiting angiogenesis of BCa by regulating phosphatase and tensin homolog deleted on chromosome ten (PTEN)/protein kinase B (Akt)/vascular endothelial growth factor A (VEGF-A) signaling pathway. Methods: Totally 62 pairs of tumor tissues and normal adjacent tissues (NAT) were collected from BCa patients who underwent surgery at Sun Yat-sen Memorial Hospital from 2016-8-2 to 2018-7-26.The expression of circNDUFA10 in tissue samples and SV-HUC-1, T24, UM-UC-3, 5637 cells were analyzed by qRT-PCR.T24 and UM-UC-3 cells were transfected with siRNA to silence siRNA, and grouped as followed:si-NC, si-circNDUFA10#1, si-circNDUFA10#2, respectively. The effect of circNDUFA10 on angiogenesis were performed by tuber formation assays and Transwell assays.The relationship of circNDUFA10 and miR-20b-5p were detected by RNA pull-down assays.T24 and UM-UC-3 cells were transfected with inhibitor and siRNA, and grouped as followed:NC inhibitor, miR-20b-5p inhibitor and miR-20b-5p inhibitor+si-circNDUFA10, respectively. The effect of miR-20b-5p on the expression of PTEN were identified by qRT-PCR.The protein expression of PTEN, Akt and p-Akt were detected by Western blot.qRT-PCR and ELISA assays were employed to detected the expression of VEGF-A induced by circNDUFA10 silencing. Results: The relative expression level of circNDUFA10 in BCa tissues (-2.450±0.192) was lower than that in paired NAT (0±0.212), t=8.576, P<0.001.The relative expression level of circNDUFA10 in SV-HUC-1(1.000±0.060) was higher than that in T24 (0.332±0.017), UM-UC-3 (0.359±0.021) and 5 637 (0.422±0.024), F=80.605, P<0.001.Compared with T24 si-NC group (1.000±0.026) and UM-UC-3 si-NC group (1.000±0.023), the relative tube formation length of HUVECs induced by BCa cells in T24 si-circNDUFA10#1 group (3.265±0.100), si-circNDUFA10#2 group (3.227±0.068) and UM-UC-3 si-circNDUFA10#1 group (2.812±0.089), si-circNDUFA10#2 group (2.760±0.056) were higher after silencing circNDUFA10, F values were 326.820 and 275.671, all P<0.001.Consistently, Transwell assays showed that silencing circNDUFA10 increased the ability of T24 and UM-UC-3 cells to promote the migration of HUVECs.Pull-down assays found that compared with T24 oligo probe group (1.000±0.038) and UM-UC-3 oligo probe group (1.000±0.052), the relative enriched level of miR-20b-5p in T24 circNDUFA10 probe group (2.836±0.093) and UM-UC-3 oligo probe group (3.354±0.136) were increased, t values were 18.31 and 16.20, all P<0.001.Rescue assays showed that si-circNDUFA10 partially reversed the effect of miR-20b-5p inhibitor on regulating the expression of PTEN.Western blot assays showed that silencing circDNUFA10 inhibited the expression of PTEN and the promoted phosphorylation of Akt.qRT-PCR showed that compared with T24 si-NC group (1.000±0.042) and UM-UC-3 si-NC group (1.000±0.037), the relative expression of VEGF-A mRNA in T24 si-circNDUFA10#1 group (1.996±0.066), si-circNDUFA10#2 group (1.949±0.082) and UM-UC-3 si-circNDUFA10#1 group (1.886±0.057), si-circNDUFA10#2 group (2.074±0.058) were increased, F values were 73.784 and 124.741, all P<0.001.Consistently, ELISA assays showed that silencing circNDUFA10 facilitated the secretion of VEGF-A. Conclusion: circNDUFA10 is lowly expressed in BCa tissues and cells, which inhibits angiogenesis of bladder cancer via miR-20b-5p/PTEN/VEGF-A axis. © 2022, Editorial Board of Chinese Journal of Cancer Prevention and Treatment. All right reserved.