Animal production units produce and store many contaminants on-site, including organic dust (OD) and hydrogen sulfide (H2S). Workers in these settings report various respiratory disease symptoms. Both OD and H2S have shown to induce lung inflammation. However, impact of co-exposure to both H2S and OD has not been investigated. Therefore, we tested a hypothesis that pre-exposure to H2S modulates the innate inflammatory response of the lungs to organic dust. In a mouse model of H2S and organic dust extract (ODE) exposure, we assessed lung inflammation quantitatively. We exposed human airway epithelial and monocytic cells to medium or H2S alone or H2S followed by ODE and measured cell viability, oxidative stress, and other markers of inflammation. Exposure to 10 ppm H2S followed by ODE increased the lavage fluid leukocytes. However, exposure to 10 ppm H2S alone resulted in changes in tight junction proteins, an increase in mRNA levels of tlr2 and tlr4 as well as ncf1, ncf4, hif1α, and nrf2. H2S alone or H2S and ODE exposure decreased cell viability and increased reactive nitrogen species production. ODE exposure increased the transcripts of tlr2 and tlr4 in both in vitro and in vivo models, whereas increased nfkbp65 transcripts following exposure to ODE and H2S was seen only in in vitro model. H2S alone and H2S followed by ODE exposure increased the levels of IL-1β. We conclude that pre-exposure to H2S modulates lung innate inflammatory response to ODE.