Hydrogen sulfide (H₂S), a metabolic end product of sulfate‐reducing bacteria, represents a genotoxic insult to the colonic epithelium, which may also be linked with chronic disorders such as ulcerative colitis and colorectal cancer. This study defined the early (30 min) and late (4 hr) response of nontransformed human intestinal epithelial cells (FHs 74 Int) to H₂S. The genotoxicity of H₂S was measured using the single‐cell gel electrophoresis (comet) assay. Changes in gene expression were analyzed after exposure to a genotoxic, but not cytotoxic, concentration of H₂S (500 μM H₂S) using pathway‐specific quantitative RT‐PCR gene arrays. H₂S was genotoxic in a concentration range from 250 to 2,000 μM, which is similar to concentrations found in the large intestine. Significant changes in gene expression were predominantly observed at 4 hr, with the greatest responses by PTGS2 (COX‐2; 7.92‐fold upregulated) and WNT2 (7.08‐fold downregulated). COX‐2 was the only gene upregulated at both 30 min and 4 hr. Overall, the study demonstrates that H₂S modulates the expression of genes involved in cell‐cycle progression and triggers both inflammatory and DNA repair responses. This study confirms the genotoxic properties of H₂S in nontransformed human intestinal epithelial cells and identifies functional pathways by which this bacterial metabolite may perturb cellular homeostasis and contribute to the onset of chronic intestinal disorders. Environ. Mol. Mutagen. 2010. © 2010 Wiley‐Liss, Inc.