Norcantharidin is the demethylated analog of cantharidin isolated from blister beetles (Mylabris phalerata Pall.). In this study, we evaluated whether norcantharidin exhibits anticancer effects against the human non-small cell lung cancer cell lines A549 (epidermal growth factor receptor (EGFR) mutation-negative) and PC9 (EGFR mutation-positive). Our results revealed that norcantharidin dose-dependently retards cell growth, arrests cell cycle at G2/M phase, reduces cell migration, and even induces apoptosis at the concentration of 100 µM. Moreover, we found that norcantharidin enhances the anticancer effects of gefitinib and cisplatin. Norcantharidin exhibited similar potency of anticancer effects against the two cell lines with different EGFR mutation status and did not affect EGF-induced EGFR phosphorylation, suggesting that the EGFR signaling may not be the target of norcantharidin. In conclusion, our results suggest that norcantharidin exhibits anticancer effects against non-small cell lung cancer cells in vitro and support its potential as a chemotherapeutic agent for treating non-small cell lung cancer.