High blood glucose commonly occurs in patients with diabetes mellitus, but little is known of its effects on intestinal epithelial cells, or its associated mechanisms of action therein. In the present study, intestinal epithelial cells were assigned to five groups: i) The normal glucose (NG) group, incubated in 5.0 mmol/l glucose; ii) the constant high glucose (CHG) group, treated with 25.0 mmol/l glucose; iii) the intermittent high glucose (IHG) group, treated with alternating doses of 5.0 and 25.0 mmol/l glucose every 8 h; iv) the mannose group, cultured in 25.0 mmol/l mannose (the osmotic control); and v) the IHG glucose + GKT137831 group, pretreated with 100 nmol/l NADPH oxidase 4 (NOX4) inhibitor, GKT137831, and then exposed to IHG. TNF-α, IL-1 and IL-6 levels were quantified using ELISA kits. Intestinal epithelial cell apoptosis was assessed by flow cytometry and oxidative stress was evaluated by reactive oxygen species (ROS) and malondialdehyde (MDA) detection. The expression levels of proteins associated with apoptosis and involved in the signal transduction of Janus kinase (JAK)/STAT3 pathway were assessed using western blot analysis. The results indicated that NOX4 expression was significantly higher in the CHG group than in the NG group (P<0.01), but lower than in the IHG group (P<0.001). The IHG group exhibited apoptosis and oxidative stress accompanied by the most significant increase in MDA, ROS and inflammatory cytokine levels (P<0.001), which was followed by that of the CHG group. Additionally, the IHG group exhibited reduced Bcl-2, as well as enhanced Bax and cleaved caspase-3 levels compared with the CHG group (P<0.001). Furthermore, the level of phosphorylated (p-)JAK/p-STAT3 was increased to a greater extent in the IHG group than in the CHG group (P<0.001). In conclusion, the findings of the present study indicated that CHG may trigger intestinal epithelial cell apoptosis and inflammation through the NOX4/ROS/JAK/STAT3 pathway, which may be aggravated by acute glucose fluctuation.