The link between per- and polyfluoroalkyl substances (PFAS) and impaired sperm quality has been disputable, partially attributed to the relatively small sample size and the lack of knowledge on exposure status in the microenvironment of target site. Here, we quantified 30 legacy and emerging PFAS in semen samples from 1206 men and enquired on their habitual nutritional supplementation. Eleven contaminant congeners were detected in over 50 % of the semen samples, topped by two legacy congeners PFOA and PFOS (median: PFOA = 0.191 and PFOS = 0.249 ng/mL). Logistic regression identified significantly positive associations between seminal PFAS and asthenospermia for PFOA, PFOS, PFHxS, and 6:2 Cl-PFESA (all P < 0.05). Quantile-based g-computation (qgcomp) model demonstrated positive overall relationship between PFAS mixtures and asthenospermia, predominantly contributed by 6:2 Cl-PFESA (qgcomp weight: 0.308) and PFHxS (qgcomp weight: 0.293), consistent with the results of generalized weighted quantile sum model, calling for more research on these PFAS replacements. Subgroup interaction analysis indicated that nutritional supplementation, primarily containing antioxidants, reduced the risk of asthenospermia and oligoasthenospermia associated with PFAS exposure, showing a protective effect. Our work demonstrated that exposure to PFOS/PFOA and their chemical replacements in semen may impact profoundly on sperm quality, mostly contributed by the early replacements 6:2 Cl-PFESA and PFHxS, and the effect of which may be reduced by active nutritional supplementation strategy for the general public.