Transmembrane protein 158 (TMEM158) plays pivotal roles in many cancers, including colorectal cancer (CRC). It has been reported that it is a recently identified upregulated gene during Ras-induced senescence. However, the clinical significance and biological functions of TMEM158 in CRC remain largely unknown. In this study, we found that TMEM158 was highly expressed in CRC tissues and cell lines compared with the corresponding noncancerous samples and normal colon epithelial cells. In vitro studies showed that TMEM158 silencing inhibited proliferation, and migration and increased apoptosis of CRC cells, whereas overexpression of TMEM158 increased proliferation, migration, and apoptosis escape of CRC cells. Mechanically, the levels of drug resistance-associated molecules, including multidrug resistance 1 and multidrug resistance protein 1, as well as the expression of antiapoptotic Bcl-2 were significantly upregulated. In addition, TMEM158 knockdown significantly inhibited tumor growth in vivo. Collectively, these results demonstrated that TMEM158 is a significant regulator of tumorigenesis and drug resistance in CRC and provided evidence that TMEM158 may be a promising target for CRC therapy.