Hyperoside (HY) is a major pharmacologically active component from Prunella vulgaris L. and Hypericum perforatum. The present study aimed to determine the anticancer effect of HY and determine the underlying mechanisms involved. Human A549 cells were treated with HY (10, 50 and 100 µM), and cell viability was detected by an MTT assay. Cell apoptosis and mitochondrial membrane potential were determined by flow cytometry. Western blot analysis was used to identify the expression of apoptosis‑associated proteins and phosphorylation of MAPK. The present study demonstrated that HY significantly inhibited the viability of A549 cells in a time‑ and dose‑dependent manner, and enhanced the percentage of apoptotic cells. HY also significantly increased the protein phosphorylation of p38 mitogen‑activated protein kinase (MAPK) and c‑Jun N‑terminal kinase (JNK), disrupted mitochondrial membrane penetrability, and triggered the release of mitochondrial cytochrome c and apoptosis‑inducing factor into the cytosol. Treatment with HY also activated the expression of caspase‑9 and caspase‑3. These results suggested that HY‑induced apoptosis was associated with activation of the p38 MAPK‑ and JNK‑induced mitochondrial death pathway. HY may offer potential for clinical applications in treating human non‑small cell lung cancer and improving cancer chemotherapy.