Clinically, one of the principal factors in the failure of advanced colorectal cancer (CRC) treatment is chemoresistance to 5-fluorouracil (5FU)-based chemotherapy. Although microRNA-375-3p (miR-375) is considered a tumor suppressor in multiple cancers, the mechanism of miR-375 in the regulation of drug resistance in CRC remains unclear. In this study, we investigated the chemosensitivity of miR-375 to 5FU in CRC from biological and clinical aspects. We found that miR-375 was significantly downregulated in CRC tissues and cell lines, and low miR-375 expression was strongly correlated with poor overall survival in CRC patients. Overexpression of miR-375 sensitized CRC cells to a broad spectrum of chemotherapeutic drugs in vitro and in vivo. Further mechanistic analysis demonstrated that miR-375 enhanced CRC cell sensitivity to 5FU by directly targeting YAP1 and SP1. MiR-375 downregulated YAP1, resulting in reduced expression of the Hippo-YAP1 pathway downstream genes CTGF, cyclin D1 and BIRC5 (also known as survivin). Overall, miR-375 was confirmed as a prospective molecular biomarker in the chemoresistance and prognosis of CRC patients, and the synergy between miR-375 and chemotherapeutic drugs could be a promising therapeutic strategy for CRC patients, especially with chemoresistance.