Recently, we identified procyanidin B2 3,3(″)-di-O-gallate (B2G2) as most active constituent of grape seed extract (GSE) for efficacy against prostate cancer (PCa). Isolating large quantities of B2G2 from total GSE is labor intensive and expensive, thereby limiting both efficacy and mechanistic studies with this novel anticancer agent. Accordingly, here we synthesized gram-scale quantities of B2G2, compared it with B2G2 isolated from GSE for possible equivalent biological activity and conducted mechanistic studies. Both B2G2 preparations inhibited cell growth, decreased clonogenicity, and induced cell cycle arrest and apoptotic death, comparable to each other, in various human PCa cell lines. Mechanistic studies focusing on transcription factors involved in apoptotic and survival pathways revealed that B2G2 significantly inhibits NF-κB and activator protein1 (AP1) transcriptional activity and nuclear translocation of signal transducer and activator of transcription3 (Stat3) in PCa cell lines, irrespective of their functional androgen receptor status. B2G2 also decreased survivin expression which is regulated by NF-κB, AP1, and Stat3 and increased cleaved PARP level. In summary, we report B2G2 chemical synthesis at gram-quantity with equivalent biological efficacy against human PCa cell lines and same molecular targeting profiles at key transcription factors level. The synthetic B2G2 will stimulate more research on prostate and possibly other malignancies in preclinical models and clinical translation.