This study investigated the effects of chlorpyrifos (CPF) on immune-cell populations and intestinal inflammation using a mouse model of inflammatory bowel disease induced by dextran sulfate sodium (DSS). C57BL/6 mice were randomly assigned to five groups with one normal control (NC) and four DSS-treated groups. Mice in the NC group were given distilled water, whereas the DSS-treated groups received distilled water containing 3% DSS for 6 days to induce colitis. The NC and disease control (DC) groups were fed a control semipurified diet, while the remaining groups were exposed to CPF in the AIN-93 diet at doses of 1, 2.5, or 5 mg/kg/day throughout the study. Results showed that dietary exposure to CPF in colitic mice significantly increased circulating classical monocytes and upregulated gene expressions of chemokines in the colon compared to the NC group. Meanwhile, CPF exposure groups had lower plasma cholinesterase activities and higher percentages of circulating neutrophils than those of the DC group. A shorten length, tissue edema, and lipid peroxidation of the colon were also observed in all CPF-exposed mice. These findings suggest that dietary exposure to CPF affected immune-cell populations and inflammatory responses, which led to more severe tissue injury in mice with DSS-induced colitis.