AbstractProstate cancer is the second most commonly diagnosed malignancy among men worldwide. Recurrently mutated in primary and metastatic prostate tumors, FOXA1 encodes a pioneer transcription factor involved in disease onset and progression through both androgen receptor-dependent and androgen receptor-independent mechanisms. Despite its oncogenic properties however, the regulation of FOXA1 expression remains unknown. Here, we identify a set of six cis-regulatory elements in the FOXA1 regulatory plexus harboring somatic single-nucleotide variants in primary prostate tumors. We find that deletion and repression of these cis-regulatory elements significantly decreases FOXA1 expression and prostate cancer cell growth. Six of the ten single-nucleotide variants mapping to FOXA1 regulatory plexus significantly alter the transactivation potential of cis-regulatory elements by modulating the binding of transcription factors. Collectively, our results identify cis-regulatory elements within the FOXA1 plexus mutated in primary prostate tumors as potential targets for therapeutic intervention.