Abstract Androgen deprivation therapy has led to elevated cases of androgen receptor (AR) pathway–independent prostate cancer with dysregulated fatty acid metabolism. However, it is unclear how prostate cancer cells sustain dysregulated fatty acid metabolism to drive AR-independent prostate cancer. Long-chain acyl-CoA synthetases (ACSL) catalyze the conversion of fatty acids into fatty acyl-CoAs that are required for fatty acid metabolism. In this study, we demonstrate that expression levels of ACSL3 and 4 were oppositely regulated by androgen–AR signaling in prostate cancer cells. AR served as a transcription suppressor to bind at the ACSL4 promoter region and inhibited its transcription. Inhibition of androgen–AR signaling significantly downregulated ACSL3 and PSA, but elevated ACSL4 levels. ACSL4 regulated a broad spectrum of fatty acyl-CoA levels, and its catalytic efficiency in fatty acyl-CoAs biosynthesis was about 1.9- to 4.3-fold higher than ACSL3. In addition, in contrast to ACSL3, ACSL4 significantly regulated global protein myristoylation or myristoylation of Src kinase in prostate cancer cells. Knockdown of ACSL4 inhibited the proliferation, migration, invasion, and xenograft growth of AR-independent prostate cancer cells. Our results suggest that the surge of ACSL4 levels by targeting AR signaling increases fatty acyl-CoAs biosynthesis and protein myristoylation, indicating the opposite, yet complementary or Yin-Yang regulation of ACSL3 and 4 levels in sustaining fatty acid metabolism when targeting androgen–AR signaling. This study reveals a mechanistic understanding of ACSL4 as a potential therapeutic target for treatment of AR-independent prostate cancer. Implications: AR coordinately regulates the expression of ACSL3 and ACSL4, such that AR pathway–independent prostate tumors become dependent on ACSL4-mediated fatty acid metabolism.