Abstract: Objective To investigate the molecular mechanism of aprepitant (Apr) reversing 5-Fluorouracil (5-FU) resistance in colorectal cancer (CRC) mouse model through endoplasmic reticulum stress (ERS). Methods Thirty mice were selected as experimental animals. Five mice were randomly assigned to control group, and the remaining 25 mice underwent subcutaneous injection in the back to establish the HCT-116/5-FU CRC mouse model. These mice were then divided into the CRC group, 5-FU group, Apr group, Apr+5-FU group and Apr+ERS inhibitor Tauroursodeoxycholic acid (TUDCA) group, with five mice in each group. Changes in body weight and tumorigenesis in mice were recorded, and their organ indicators were calculated. Western blotting (WB) was used to detect the protein expression levels of protein kinase R-like ER kinase (PERK), eukaryotic initiation factor 2 subunit α (eIF2α),activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) in each group. Results At 5, 10, 15 and 20 d after medication, there were no statistically significant differences in body weight among CRC, 5-FU, Apr, Apr+5-FU and Apr+TUDCA groups, neither in time points nor in interactions (P>0.05). Two days after the last administration, there was no significant difference in the indexes of thymus, lung, liver, spleen, heart, kidney and other organs among all groups (P>0.05). Compared with CRC group, the protein expression levels of PERK, P-EIF2α/eIF2α, ATF4 and CHOP in Apr group and Apr+5-FU group were significantly increased, the number of tumors was significantly decreased, the tumor mass was significantly decreased, and the tumor volume was significantly decreased (P<0.05), and the improvement of Apr+5-FU group was better than that of other groups (P<0.05). Conclusion Apr can enhance chemotherapy sensitivity and reverse chemotherapy resistance in CRC mice, which may be mediated by downstream molecules of ERS pathway.