Red onion skin waste (ROSW) was analyzed for extraction of naturally occurring 4′-O-glucoside of quercetin, spiraeoside (SPI) with promising biological activities. Reversed-phase high-performance liquid chromatography was used to determine the SPI content in three different solvent extracts of ROSW: water (12.2 mg/g), methanol (27.6 mg/g), and ethanol (32.5 mg/g). The ethanol extract and SPI showed significant radical-scavenging and anti-inflammatory activities. In addition, the anti-cancer effects of SPI on a HeLa cells was investigated. The results indicated that SPI treatment significantly inhibited cell growth, and the dose of 50 μg/mL exhibited the highest anti-cancer activity. SPI inhibited the expression of B-cell lymphoma 2 and BH3-interacting domain-death agonist and promoted apoptosis by activating caspase-9/-3 expression. Notably, SPI inhibited the expression of mu-2-related death-inducing gene, a molecule involved in death receptor-mediated apoptotic signaling. Cyclin-dependent kinase 2-cyclin-E expression was also inhibited after SPI treatment, particularly at the G2/M checkpoint. Our findings provide novel insights into the apoptotic potential with promising anticancer and enzyme inhibitory effects of ROSW SPI. Display omitted •Spiraeoside is a naturally occurring quercetin glucoside extracted from red onion skin.•Red onion skin and spiraeoside showed potential antioxidant and anti-inflammatory effects.•Red onion skin spiraeoside showed promising anti-cancer effect against HeLa cell.•Spiraeoside provided novel insights on apoptotic potential and enzyme inhibitory effects.