Colorectal cancer (CRC) is the second leading cause of cancer-related illness worldwide and one of the most common malignancies. Therefore, colorectal cancer research and cases have gained increasing attention. Oxaliplatin (OXA) is currently used in first-line chemotherapy to treat stage III and stage IV metastatic CRC. However, patients undergoing chemotherapy often develop resistance to chemo drugs being used. Evidence has confirmed that microRNAs regulate downstream genes in cancer biology and thereby have roles related to tumor growth, proliferation, invasion, angiogenesis, and multi-drug resistance. The aim of our study is to establish whether miR-31-5p is an oncogene in human colorectal cancers that are resistant to OXA and further confirm its malignant phenotype-associated target molecule. From the results of miRNA microarray assay, we establish that miR-31-5p expression was upregulated in oxaliplatin-resistant (OR)-LoVo cells compared with parental LoVo cells. Moreover, through in vitro and in vivo experiments, we demonstrate that miR-31-5p and large tumor suppressor kinase 2 (LATS2) were inversely related and that miR-31-5p and Forkhead box C1 (FOXC1) were positively correlated in the same LoVo or OR-LoVo cells. Importantly, we reveal a novel drug-resistance mechanism in which the transcription factor FOXC1 binds to the miR-31 promoter to increase the expression of miR31-5p and regulate LATS2 expression, resulting in cancer cell resistance to OXA. These results suggest that miR-31-5p may be a novel biomarker involved in drug resistance progression in CRC patients. Moreover, the FOXC1/miR31-5p/LATS2 drug-resistance mechanism provides new treatment strategies for CRC in clinical trials.