Perfluoroalkyl ether carboxylic acids (PFECA) represent an important group of emerging per- and polyfluoroalkyl substances (PFAS). PFECA have been found in a variety of environmental matrices. While legacy PFAS have been extensively documented for their reproductive toxicity, the health implications of emerging alternatives like PFECA remains unclear. The present study is aimed at investigating the potential reproductive toxicity of perfluoro-(3,5,7,9-tetraoxadecanoic) acid (PFO4DA), a type of PFECA, through combining in vivo and in vitro assays. Adult male BALB/c mice were exposed to 2 and 10 mg/kg body-weight/day of PFO4DA via oral gavage for four consecutive weeks. While no overt structural or morphological changes were observed in the testes following PFO4DA exposure, significant reductions in both sperm quantity and quality were evident, correlating with increased PFO4DA dosage. PFO4DA exposure compromised the integrity of the blood-testis barrier (BTB) and led to a moderate decrease in BTB-associated junction proteins, including Connexin-43 and Claudin-11. Notably, PFO4DA disrupted androgen production, resulting in a significant reduction in testosterone (T) levels (>
60 %) in both serum and testicular tissue. Mechanistic analysis revealed that PFO4DA significantly repressed the expression levels of steroidogenic genes StAR and CYP11A1 both in testes and in cultured Leydig cells (TM3). These findings suggest that PFO4DA primarily targets the steroidogenic pathway in Leydig cells by suppressing StAR and CYP11A1, thereby inhibiting T synthesis. The subsequent T deficiency impairs Sertoli cell function and BTB integrity, ultimately disrupting spermatogenesis and impacting male reproductive health.