Stearoyl-CoA desaturase (SCD), the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids, is highly expressed in prostate cancer although the SCD protein has been known to be rapidly turned over by proteolytic cleavage. The present data demonstrate that SCD can promote proliferation of androgen receptor (AR)-positive LNCaP prostate cancer cells and enhance dihydrotestosterone (DHT)-induced AR transcriptional activity, resulting in increased expression of prostate-specific antigen (PSA) and kallikrein-related peptidase 2 (KLK2). Interestingly, among the previously reported SCD-derived peptides produced by proteolytic cleavage of SCD, a peptide spanning amino acids 130-162 of SCD (SCD-CoRNR) contained the CoRNR box motif (LFLII) and enhanced AR transcriptional activity. In contrast, a mutant SCD-CoRNR in which Leu136 was replaced by Ala had no effect on AR transcriptional activity. Moreover, SCD-CoRNR directly interacted with AR and inhibited RIP140 suppression of AR transactivation. Knockdown of the SCD gene by SCD microRNA suppressed AR transactivation with decreased cell proliferation, suggesting that SCD may regulate the proliferation of LNCaP cells via modulation of AR transcriptional activity. Moreover, ectopic expression of SCD in LNCaP cells facilitated LNCaP tumor formation and growth in nude mice. Together, the data indicate that SCD plays a key role in the regulation of AR transcriptional activity in prostate cancer cells.