Scyreprocin is an antimicrobial peptide first identified in the mud crab Scylla paramamosain. Herein, we showed that its recombinant product (rScyreprocin) could significantly inhibit the growth of human lung cancer NCI-H460 cells (H460), but showed no cytotoxicity to human lung fibroblasts (HFL1). rScyreprocin was a membrane-active peptide that firstly induced the generation of reactive oxygen species (ROS) in H460, and led to endoplasmic reticulum stress and Ca2+ release, which resulted in mitochondrial dysfunction and subsequently activation of caspase-3 cascades, and ultimately led to apoptosis. The comprehensive results indicated that rScyreprocin exerted anticancer activity by disrupting cell membrane and inducing apoptosis. The in vivo efficacy test demonstrated that intratumoral injection of rScyreprocin significantly inhibited the growth of H460 xenografts, which was close to that of the cisplatin (inhibition rate: 69.94% vs. 80.76%). Therefore, rScyreprocin is expected to become a promising candidate for the treatment of lung cancer.