The present study aims to investigate the roles of U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) in the resistance to anti-androgen treatment in prostate cancer and its underlying mechanism. U2AF1 and androgen receptor variant 7 (ARV7) knockdown and overexpression were introduced in PC3 and DU145 cells. In addition, a bicalutamide-resistant PC3 (PC3 BR) cell line was also constructed. Cell count, MTT and soft agar colony formation assays were performed to evaluate cell proliferation. qRT-PCR was applied to determine the mRNA levels of U2AF1, ARV7 and Mitogen-Activated Protein Kinase 1 (MAPK1). Western blot was used to determine the MAPK1 protein expression. A negative correlation between ARV7 and U2AF1 in prostate tumor tissues was observed. U2AF1 downregulation was correlated with poor prognosis in prostate cancer patients. U2AF1 exhibited a negative correlation with ARV7 and its downregulation promoted prostate cancer cell proliferation and bicalutamide resistance. The regulatory effects of U2AF1 on ARV7 splicing were associated with MAPK1. U2AF1 affected prostate cancer proliferation and anti-androgen resistance by regulating ARV7 splicing. (C) 2021 Elsevier Inc. All rights reserved.