Background: Tumor cells can evade immune surveillance and immune killing effects during neo-endocrine therapy resistance, but the mechanisms that induce this phenomenon are still unclear. Flightless I homolog (FLII) functions as a coregulator of transcription factors in malignancies. Here, we aim to identify the functions and mechanisms of FLII-mediated neo-endocrine therapy resistance and immune evasion in prostate cancer (PCa).Methods: Bioinformatics analysis and PCa tissue microarray were used to assess the correlation between FLII and the immune checkpoint PD-L1. Immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) were used to investigate the regulation of PD-L1 expression by FLII. Enzyme-linked immunosorbent assay (ELISA) was used to study the effect of FLII on cytokine secretion. The number of effector T cells and immunosuppressive lymphocytes was detected by flow cytometry (FCM).Results: FLII was negatively correlated with the expression of PD-L1 in PCa. Mechanistic studies demonstrated that FLII physically interacted with YBX1 to inhibit the nuclear localization of YBX1 and thereby suppressed the transcriptional expression of PD-L1 in enzalutamide-resistant tumors. Restoration of FLII expression reversed neo-endocrine therapy resistance by activating the T cell response in the tumor microenvironment mediated by partial inhibition of the YBX1/PD-L1 pathway. The reversal of neo-endocrine therapy resistance and immune evasion was mediated by the proliferation of effector CD8+ T cells and inhibition of the infiltration of regulatory T cells and myeloid-derived suppressor cells in the tumor microenvironment.Conclusions: FLII inhibits tumor progression in part through repressing YBX1/PD-L1-mediated immune evasion. Combination therapy with FLII and neo-endocrine therapy may improve the benefits in prostate cancer patients and avoid immune evasion.