5-Fluorouracil (5-FU)-based chemotherapy is the first-line option for patients with advanced colorectal cancer (CRC). However, the development of chemoresistance is the primary cause of treatment failure. Halofuginone (HF), a small molecule alkaloid derived from febrifugine, has been demonstrated to exert strong anti-proliferative effects. However, to the best of our knowledge, whether HF inhibits the progression of 5-FU-resistant human CRC HCT-15/FU cells, and the underlying mechanisms, remain unknown. In the present study, the effects of HF on HCT-15/FU cells were assessed in vitro. The results revealed that HF inhibited HCT-15/FU cell viability as demonstrated by the MTT and colony formation assays. Following treatment of HCT-15/FU cells with HF, the migratory and invasive capacities of the cells were significantly decreased. MicroRNA (miRNA/miR)-sequencing data, subsequent miRNA trend analysis and reverse transcription-quantitative PCR all demonstrated that miR-132-3p expression was increased following treatment with HF in a dose-dependent manner. Western blot analysis indicated that following treatment with HF, the expression levels of proteins associated with proliferation, invasion and metastasis in cells were markedly downregulated. These results suggested that HF inhibited the proliferation, invasion and migration of HCT-15/FU cells by upregulating the expression levels of miR-132-3p. Therefore, miR-132-3p may serve as a molecular marker, which may be used to predict CRC resistance to 5-FU, and HF may serve as a novel clinical treatment for 5-FU-resistant CRC. © 2020 Spandidos Publications. All rights reserved.