Tumor suppressor candidate 3 (TUSC3) is a coding gene responsible for N-glycosylation of many critical proteins. TUSC3 gene plays an oncogenic role in colorectal cancer (CRC), however, the role of TUSC3 in drug resistance of CRC is still unclear. The aim of this study is to investigate the biological function and molecular mechanism of TUSC3 in CRC drug resistance. The expression of TUSC3 in CRC is positively correlated to tumor stage in 90 paired clinical samples, and negatively associated with overall survival and disease-free survival of CRC patients. In vitro, TUSC3 promotes the formation of stemness and induces the drug resistance to 5-fluorouracil (5-FU) and cis-Dichlorodiammineplatinum(II) (DDP) in CRC cells. The tissue microarray assay and bioinformatic analysis indicates that TUSC3 may promote the expression of CD133 and ABCC1 via hedgehog signaling pathway. Treatment of Hedgehog signaling pathway agonist or inhibitor in TUSC3-silenced or TUSC3-overexpressed cells reverse the effects of TUSC3 in cellular stemness phenotype and drug resistance. Meanwhile, co-immunoprecipitation and immunoﬂuorescence assays indicate a tight relationship between TUSC3 and SMO protein. Our data suggests that TUSC3 promotes the formation of cellular stemness and induces drug resistance via Hedgehog signaling pathway in CRC.© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: email@example.com.