Rubus chingii is a traditional Chinese medicinal herbal that has been used since ancient times for its great dietary and medicinal values. Recent reports have underscored the promising cytotoxic effect of R. chingii extracts against a wide variety of cancer cells. Therefore, in the current study, we aim to explore the anticancer potential of the Rubus chingii ethanolic leaf extract (RcL-EtOH) against non-small cell lung cancer A549 cells. RcL-EtOH efficiently exerted a cytotoxic effect against A549 cells in a dose dependent manner, whilst, it exhibited non-significant toxic effects on normal murine macrophage cells, signifying its safety against normal cells. The reduced viability of A549 cells was reaffirmed by the acridine orange/ethidium bromide double staining, which confirmed the induction of apoptosis in RcL-EtOH-treated A549 cells. In addition, RcL-EtOH instigated the dissipation of mitochondrial membrane potential (DYm) with mutual escalation in ROS generation in a dose-dependent manner. Furthermore, RcL-EtOH increased caspase-3, caspase-9 levels in A549 cells post-exposure to RcL-EtOH, which was concomitantly followed by altered mRNA expression of apoptotic (anti-apoptotic: Bcl-2, BclXL; pro-apoptotic: Bax, Bad). To sum up, the RcL-EtOH-instigated apoptotic cell death within A549 cells was assumed to be accomplished via targeting mitochondria, triggering increased ROS generation, with subsequent activation of caspase cascade and altering the expression of gene regulating apoptosis. Collectively, RcL-EtOH might represent a plausible therapeutic option for the management of lung cancer.