Perfluorooctanesulfonic acid (PFOS) is a widely recognized environment pollutant known for its high bioaccumulation potential and a long elimination half-life. Several studies have shown that PFOS can alter multiple biological pathways and negatively affect human health. Considering the direct exposure to the gastrointestinal (GI) tract to environmental pollutants, PFOS can potentially disrupt intestinal homeostasis. However, there is limited knowledge about the effect of PFOS exposure on normal intestinal tissues, and its contribution to GI-associated diseases remains to be determined. In this study, we examined the effect of PFOS exposure on the gene expression profile of intestinal tissues of C57BL/6 mice using RNAseq analysis. We found that PFOS exposure in drinking water significantly downregulates mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme, in intestinal tissues of mice. We found that diets containing the soluble fibers inulin and pectin, which are known to be protective against PFOS exposure, were ineffective in reversing the downregulation of HMGCS2 expression in vivo. Analysis of intestinal tissues also demonstrated that PFOS exposure leads to upregulation of proteins implicated in colorectal carcinogenesis, including β-catenin, c-MYC, mTOR and FASN. Consistent with the in vivo results, PFOS exposure leads to downregulation of HMGCS2 in mouse and human normal intestinal organoids in vitro. Furthermore, we show that shRNA-mediated knockdown of HMGCS2 in a human normal intestinal cell line resulted in increased cell proliferation and upregulation of key proliferation-associated proteins such as cyclin D, survivin, ERK1/2 and AKT, along with an increase in lipid accumulation. In summary, our results suggest that PFOS exposure may contribute to pathological changes in normal intestinal cells via downregulation of HMGCS2 expression and upregulation of pro-carcinogenic signaling pathways that may increase the risk of colorectal cancer development.