Colorectal cancer (CRC) is a frequent fatal cancer worldwide. 5-Fluorouracil (5-FU) is extensively used in its chemotherapy. This drug resistance, however, should be well concerned. Ring finger proteins (RNF) are vital regulators involved in CRC development. In this article, HCT116R cells were first established. The roles of RNF38 and Wnt signaling in 5-FU-resistant CRC were further illustrated. Our study provides novel evidence for improving 5-FU chemotherapy outcome in CRC patients.The phenotype of established HCT116R cells was first examined. Next, the regulatory effect of RNF38 on 5-FU resistance in CRC was mainly explored. Nude mice bearing CRC were treated with 5-FU and in vivo overexpression of RNF38.5-FU-resistant HCT-116 cells (HCT116R) were first established. 5-FU treatment markedly killed survival and induced apoptosis in HCT-116 cells. P53 was downregulated in HCT116R cells. Through microarray analysis, RNF38 was found to be upregulated in HCT116R cells compared to parental cells.Overexpression of RNF38 enhanced 5-FU resistance in CRC. Furthermore, Wnt signaling was activated by RNF38 and involved in 5-FU resistance in CRC.