Perfluorotridecanoic acid (PFTrDA) is a long-chain (C13) perfluoroalkyl carboxylic acid. Here, we report the influence of PFTrDA exposure on the maturation of rat Leydig cells in late puberty in vivo. Male Sprague–Dawley rats were administered PFTrDA by gavage of 0, 1, 5, and 10 mg/kg/day from 35 days to 56 days postpartum. PFTrDA had no effect on body weight, testis weight, and epididymis weight. It significantly decreased the serum testosterone level after 5 and 10 mg/kg exposure, while it did not alter the serum estradiol level. The serum luteinizing hormone level was markedly reduced after 10 mg/kg PFTrDA exposure, while the follicle-stimulating hormone level was unchanged. Star, Cyp11a1, Cyp17a1, Hsd3b1, and Insl3 transcript levels in the testis were markedly lowered in the 1–5 mg/kg PFTrDA group and the Lhb transcript level in the pituitary in the 10 mg/kg group. CYP11A1 and HSD11B1-positive Leydig cell numbers were markedly reduced after 10 mg/kg PFTrDA exposure. Testicular triglyceride and free fatty acid (palmitic acid, oleic acid, and linoleic acid) levels were significantly reduced by PFTrDA, while Mgll (up-regulation) and Scarb1 and Elovl5 (down-regulation) expression were altered. AKT1 and AMPK phosphorylation was stimulated after 10 PFTrDA mg/kg exposure. In conclusion, PFTrDA delays the maturation of Leydig cells in late puberty mainly by altering the free fatty acid profile.