The present study was undertaken to determine the reproductive hazards of Di-(2-ethylhexyl)-phthalate (DEHP) on mouse spermatozoa and embryos in vitro and genomic changes in vivo. Direct low-level DEHP exposure (1 μg/ml) on spermatozoa and embryos was investigated by in vitro fertilization (IVF) process, culture of preimplanted embryos in DEHP-supplemented medium and embryo transfer to achieve full term development. Big Blue® transgenic mouse model was employed to evaluate the mutagenesis of testicular genome with in vivo exposure concentration of DEHP (500 mg/kg/day). Generally, DEHP-treated spermatozoa (1 μg/ml, 30 min) presented reduced fertilization ability (P<0.05) and the resultant embryos had decreased developmental potential compared to DMSO controls (P<0.05). Meanwhile, the transferred 2-cell stage embryos derived from treated spermatozoa also exhibited decreased birth rate than that of control (P<0.05). When fertilized oocytes or 2-cell stage embryos were recovered by in vivo fertilization (without treatment) and then exposed to DEHP, the subsequent development proceed to blastocysts was different, fertilized oocytes were significantly affected (P<0.05) whereas developmental progression of 2-cell stage embryos was similar to controls (P>0.05). Testes of the Big Blue® transgenic mice treated with DEHP for 4 weeks indicated an approximately 3-fold increase in genomic DNA mutation frequency compared with controls (P<0.05). These findings unveiled the hazardous effects of direct low-level exposure of DEHP on spermatozoa's fertilization ability as well as embryonic development, and proved that in vivo DEHP exposure posed mutagenic risks in the reproductive organ - at least in testes, are of great concern to human male reproductive health.