Wingless-type (Wnt) family of secreted glycoproteins is a group of signal molecules implicated in oncogenesis. Abnormal activation of Wnt signal pathway is associated with a variety of human cancers, including non-small cell lung cancer (NSCLC). Wnt antagonists, such as the secreted frizzled-related protein (SFRP) family, Wnt inhibitory factor-1 (WIF-1) and cerberus, inhibit Wnt signal pathway by directly binding to Wnt molecules. Norcantharidin (NCTD) is known to possess anticancer activity but less nephrotoxicity than cantharidin. In this study, we found that NCTD inhibited cell proliferation, induced apoptosis, arrested cell cycle and suppressed cell invasion/migration in vitro. Additionally, Wnt signal pathway transcription was also suppressed. NCTD treatment blocked cytoplasmic translocation of beta-catenin into the nucleus. Alterations of apoptosis-related proteins, such as Bax, cleaved caspase-3 (pro-apoptotic) and Bcl-2 (anti-apoptotic), had been detected. Furthermore, the expression levels of WIF-1 and SFRP1 were significantly increased in NCTD-treated groups compared with negative control (NC) groups. Abnormal methylation was observed in NC groups, while NCTD treatment promoted WIF-1 demethylation. The present study revealed that NCTD activated WIF-1 via promoter demethylation, inhibiting the canonical Wnt signal pathway in NSCLC, which may present a new therapeutic target in vivo.