Hydrogen sulfide gas (H2S) is a chemical weapon and a common environmental pollutant. H2S intoxication is lethal to humans and animals. H2S contact to the eye can cause vision loss. However, the molecular mechanisms associated with H2S toxicity to the cornea remain unclear, and no specific therapy exists to mitigate ocular damage from H2S. Here, we report H2S‐induced cytotoxicity and the parameters contributing to the molecular mechanisms associated with corneal toxicity using primary human corneal stromal fibroblasts (hCSFs) in vitro. Sodium hydrosulfide (NaSH) was used as a source of H2S, and the cytotoxicity of H2S was determined by treating hCSF cells with varying concentrations of NaSH (0–10 mM) for 0–72 hours. Changes in cell proliferation, oxidative stress factors, and the expression of inflammatory and fibrotic genes were studied using standard commercial kits and qRT‐PCR. NaSH exposure to hCSFs showed dose‐ and time‐dependent cytotoxicity. The IC50 of NaSH was determined to be 5.35 mM. NaSH 5.35 mM exposure led to significantly decreased cytochrome c oxidase activity, increased ROS production, and increased expression of inflammatory and fibrotic genes in hCSF cells. H2S/NaSH exposure alters normal mitochondrial function, oxidative stress, and inflammatory and fibrotic gene responses in corneal stromal fibroblasts in vitro. Hydrogen sulfide gas (H2S) is a chemical weapon and a common environmental pollutant. H2S intoxication is lethal to humans and animals. H2S contact to the eye can cause vision loss. Here, we report dose‐ and time‐dependent cytotoxicity of H2S gas exposure to human corneal stromal fibroblasts and parameters contributing to the mechanism associated with H2S toxicity to the human cornea using an in vitro model.