Phthalates are suspected endocrine disrupting chemicals that impair male reproductive function in animal and epidemiological studies. We investigated associations between urinary phthalate metabolites and acrosin activity, along with that between insulin like-factor 3 (INSL3), a Leydig cell function marker, in Chinese adult men and assessed the association between the metabolites and male reproductive function. Serum levels of INSL3 and other hormones, semen parameters, and urinary concentrations of 14 phthalate metabolites in 1066 men were measured. The unadjusted concentrations of phthalates were included as independent variables and urinary creatinine as a separate covariate. INSL3 was negatively associated with mono-2-ethylhexyl phthalate (MEHP) and %MEHP [percentage of MEHP to all di(2-ethylhexyl) phthalate (DEHP) metabolites]. Acrosin activity was negatively associated with mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), MEHP and %MEHP. MBP and MiBP were also negatively associated with total testosterone (T), free androgen index (FAI), free testosterone (FT), luteinizing hormone (LH) and sperm morphology and positively associated with DNA fragmentation index (DFI). A negative association between %MEHP and sperm motility was observed. Several other metabolites were also associated with reproductive function. This is the first report on the inverse associations of phthalate metabolites with acrosin activity and INSL3. Phthalates may cause multiple adverse results on reproductive function at environmental levels. (C) 2015 Elsevier B.V. All rights reserved.