Perfluoroalkyl chemicals induce male reproductive toxicity. Current evidence showed the effects of the chemical exposure on the deterioration of testicular functions, and reduction in epididymal sperm counts. Previous studies showed that PFOA and PFOS displayed a high correlation with each other in seminal plasma levels, but induced different effects on semen variables. In this study, we focused on the comparative toxicity analysis of PFOA and PFOS, using a rat primary Sertoli cell model. Our transcriptomic data showed that PFOA and PFOS treatments (40 μM) perturbed global gene expression. While PFOS induced higher toxicity in affecting cytoskeleton signaling, Sertoli cell-cell junction, and inflammation, underlined by Ingenuity pathway analysis. Immunocytochemical staining revealed that PFOS treatment (40 and 80 μM) induced truncated actin filament and disorganized bundled configuration in the cell cytoplasm. Moreover, disorganized distribution of N-cadherin (N-cad) and β-catenin (β-cat), and defragmentation of ZO-1 at the Sertoli cell-cell interface was evident. At 80 μM of PFOS, cytoplasmic distribution of N-cad, β-cat, and ZO-1 were observed. We then examined whether resveratrol, a polyphenol antioxidant, was able to protect the cells from PFOS toxicity. The pretreatment of Sertoli cells with 10 μM resveratrol prevented the formation of truncated actin filament and dis-localization of β-cat. Western blot analysis showed that Res pretreatment increased the levels of basal ES proteins (N-cad and β-cat), tight junction proteins (ZO-1 and occludin), and gap junction protein, versus control.