Transmembrane protein 88 (TMEM88) is a transmembrane protein that plays a crucial role in regulating human stem cell differentiation and embryonic development. However, its expression and clinicopathologic significance in human neoplasms is unclear. In this study, the expression and subcellular localizations of TMEM88 were assessed in 214 cases of non–small cell lung cancer (NSCLC). Notably, TMEM88 was highly expressed in the cytosol of 60%NSCLC specimens examined. Higher expression of cytosolic TMEM88 inNSCLC correlated significantly with poor differentiation, high TNM stage, lymph node metastasis, and inferior survival. In NSCLC cells displaying membrane-localized TMEM88, we observed an inhibition of canonical Wnt signaling due to interactions of TMEM88 with the Wnt pathway factor Dishevelled (DVLS). In contrast, NSCLC cells with cytosol-localized TMEM88 lacked effects on Wnt signaling. Cytosolic interactions of TMEM88 and DVLS increased the expression of phosphorylated, active forms of p38, GSK3b (Thr390), and Snail, thereby reducing the expression of the tight junction-associated proteins ZO-1 and occludin, effects associated with enhanced invasive and metastatic cell characters. Importantly, attenuating the expression of cytosolic TMEM88 reduced metastatic prowess in xenograft models. Overall, our findings show howmislocalization of TMEM88 to the cytosol in NSCLC cells ablates its Wnt pathway regulatory properties, thereby promoting invasion and metastasis by activating the p38–GSK3b–Snail signaling pathway. Cancer Res; 75(21); 1–11. 2015 AACR.