Cucurbitacin-B (Cur-B) is an analogue triterpenoid belonging to the Cucurbitaceae family. Previous reports have explicitly outlined various biological activities of Cucurbitaceae family members, including the anticancer activity of Cur-B. In the present study, we tried to elucidate the anticancer efficacy of Cur-B against prostate cancer PC3 cells. PC3 cells were exposed to purified Cur-B at 5, 10, 15, 20 and 25 µM for 24. Cur-B exposure reduced cell viability of PC3 cells at 5 µM (p < 0.05), with further reduction with increased Cur-B concentration (15 µM, p < 0.01 and 25 µM, p < 0.001). Cur-B also succeeded in instigating nuclear fragmentation and condensation, followed by activation of caspase-8, -9 and -3 proportionally with increasing concentrations of Cur-B. Treatment with Cur-B also instigated ROS-mediated oxidative stress both qualitatively and quantitatively at 5 µM, p < 0.05; 15 µM, p < 0.01 and 25 µM, p < 0.001. Increased ROS after Cur-B treatment also led to dissipation of mitochondrial membrane potential, thereby resulting in considerable apoptosis (p < 0.001), which, again, was proportionally dependent on Cur-B concentration. Cur-B exposure to PC3 cells was concomitantly followed by reduced cyclin D1, cyclin-dependent kinase 4 (CDK4) expression and augmented mRNA expression of CDK inhibitor p21Cip1. Intriguingly, Cur-B exposure also led to considerable downregulation of the JAK/STAT signaling cascade, which may be the reason behind Cur-B-mediated apoptosis and cell cycle arrest within PC3 cells. Therefore, these observations explicitly establish that Cur-B could serve in the prevention of prostate cancer.