Studies showed that microRNAs (miRNAs) are important regulators in drug resistance. The current study investigated the role of miR-185-3p and its predicted target gene AQP5 in 5-FU-insensitive colorectal cancer (CRC) cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and Spearman's correlation analysis were conducted to determine the correlation of expression levels of miR-185-3p and AQP5 from CRC tissues. HCT-116 and HCT-8 cells were treated by gradient concentration of 5-FU to construct 5-FU-resistant CRC model. The inhibition and viability of 5-FU-resistant cells were detected by MTT assay, and cell migration and invasion ability were determined by wound healing and transwell assay. The expressions of miR-185-3p and AQP5 were measured by qRT-PCR. StarBase and dual-luciferase reporter assay were used to predict and confirm the interaction between miR-185-3p and AQP5. Further experiments were performed to explore the function of miR-185-3p in 5-FU-resistant cells through regulating aquaporin-5 (AQP5). The levels of EMT-associated markers and AQP5 were determined by conducting Western Blot and qRT-PCR. We found that 5-FU-resistant CRC cells showed a lower inhibition rate, and higher migration and invasion abilities. MiR-185-3p was low-expressed in CRC tissues and 5-FU-resistance cells, and it targeted and regulated the expression of AQP5, which was found up-regulated in CRC and 5-FU-resistance CRC cells (r = -0.29, P < .05). Furthermore, miR-185-3p mimic enhanced the chemo-sensitivity of 5-FU-resistant cells, while overexpressed AQP5 reversed such an effect produced by miR-185-3p mimic. MiR-185-3p mimic enhances the chemosensitivity of CRC cells via AQP5. Our research provides a potential therapeutic target for 5-FU-resistant CRC cells.