Prostate cancer (PCa) is the second leading cause of cancer death in men. PCa progression can be associated with obesity. Signal transducer and activator of transcription-3 (STAT3) plays a crucial role in PCa growth. However, whether STAT3 plays a role in high-fat diet (HFD)-associated PCa growth is unknown. Our data show that HFD feeding increases tumor size, STAT3 phosphorylation, and palmitic acid (PA) level in the xenograft tissues of the PCa-bearing xenograft mouse model. In vitro studies show that PA increases STAT3 expression and phosphorylation (STAT3-Y705) in PCa. Computational modeling suggests strong and stable binding between PA and unphosphorylated STAT3 at R593 and N538. The binding changes STAT3 structure and activity. Functional studies show that both STAT3 mutants (R583A and N538A) and STAT3 dominant negative significantly reduce PA-enhanced STAT3 phosphorylation, PA-increased PCa cell proliferation, migration, and invasion. In the xenograft mouse models, the HFD-increased tumor growth and STAT3 phosphorylation in tumors are reversed by STAT3 inhibition. Our study not only demonstrates the regulatory role of PA/STAT3 axis in HFD-associated PCa growth but also suggests a novel mechanism of how STAT3 is activated by PA. Our data suggest STAT3 as a therapeutic target for the treatment of HFD-associated PCa.