Transcriptomic and LC-MS/MS-based targeted lipidomic analyses were conducted to identify the effects of in utero PFOS exposure on neonatal testes and its relation to testicular dysfunction in adult offspring. Pregnant mice were orally administered 0.3 and 3 μg PFOS/g body weight until term. Neonatal testes (P1) were collected for the detection of PFOS, and were subjected to omics study. Integrated pathway analyses using DAVID, KEGG, and IPA underlined the effects of PFOS exposure on lipid metabolism, oxidative stress and cell junction signaling in testes. LC-MS/MS analysis showed that the levels of adrenic acid and docosahexaenoic acid (DHA) in testes were significantly reduced in the PFOS treatment groups. A significant linear decreasing trend in eicosapentaenoic acid and DHA with PFOS concentrations was observed. Moreover, LOX-mediated 5-hydroxyeicosatetraenoic acids (HETE) and 15-HETE from arachidonic acid in the testes were significantly elevated and a linear increasing trend of 15-HETE concentrations was detected with doses of PFOS. The perturbations of lipid mediators suggested that PFOS has potential negative impacts on testicular functions. Postnatal analysis of male offspring at P63 showed significant reductions in serum testosterone and epididymal sperm count. This study sheds light into the as yet unrevealed action of PFOS on lipid mediators in affecting testicular functions.