Previous studies have established the in vivo bioavailability and efficacious dosages of phenylbutyl isoselenocyanate (ISC-4), a selenium-substituted isothiocyanate, against mouse xenograft models of human melanoma and colorectal cancer. To explore its potential attributes against prostate cancer, we treated human LNCaP prostate cancer cells with ISC-4 and examined their apoptosis responses, and interrogated the signaling mechanisms through pharmacological and siRNA knockdown approaches. Our results show that ISC-4 was more potent at inducing apoptosis than its sulfur analog phenylbutyl isothiocyanate (PBITC) without suppressing protein kinase AKT Ser⁴⁷³ phosphorylation. ISC-4 induced apoptosis in concentration- and time-dependent manners, and the apoptosis execution was attenuated by pre-incubation with a pan caspase inhibitor. ISC-4 decreased the abundance of androgen receptor (AR) and its best known target prostate specific antigen (PSA) without decreasing their steady state mRNA. ISC-4 upregulated the abundance of p53 protein and its Ser¹⁵ -phosphorylative activation, and that of DNA double strand break marker Ser¹³⁹ -p-H2A.X coincident with apoptotic exposure. Similar to the rapid induction of reactive oxygen species (ROS) by isothiocyanates, ISC-4 increased dihydroethidium-detectable signals in LNCaP cells. Pre-incubation with ROS scavenger N-acetyl-l-cysteine preserved AR and PSA abundance, markedly reduced ISC-4-induced apoptosis and attenuated p53 Ser phosphorylation, p21Cip1, and p-H2A.X. Furthermore, siRNA knockdown of p53 did not suppress ROS production, but decreased ISC-4-induced apoptosis. Knocking down p53-targets PUMA and Bax exerted greater protective effect on ISC-4-induced apoptosis than depleting p21Cip1. In summary, ISC-4 inhibited LNCaP cell growth and survival with ROS-mediated suppression of AR axis signaling and induction of p53-PUMA-Bax mitochondrial apoptosis.