BACKGROUND: Bovine viral diarrhea virus (BVDV) is one of the most important pathogens. OBJECTIVES: The aim of this study was to investigate the effects of cytopathic (CP) and non-cytopathic (NCP) biotypes of BVDV on vital status, membrane integrity, and motility of sperm cells in Holstein dairy bulls in vitro. METHODS: BVDV-free frozen semen samples were counted after thawing and centrifuged to separate live sperms. A sample containing 105 spermatozoa/mL was prepared. CP and NCP BVDV with 3 different doses of 105 (high dose), 104 (medium dose), and 103 (low dose) tissue culture infectious dose (TCID) 50/mL were challenged to sperm cells. After 2 hours of incubation at 38.5°C, eosin-nigrosine staining and hypoosmotic swelling (HOS) test were performed to assess the sperm viability and plasma membrane integrity. Computer assisted semen analysis (CASA) was used to evaluate the sperm motility parameters. The obtained data were analyzed using GLM method in SAS software. RESULTS: The percentage of live spermatozoa in the control group was 72±3.60%. However, it decreased significantly with the increase of virus concentration in both groups (p ≤0.05). Sperm integrity in the control group showed that the quality of semen was 65± 3.21. But the effect of virus biotypes resulted in a significant decrease in both high (105) and low (103) concentrations (p ≤0.05). BVDV biotypes are able to reduce different sperm movements as their concentration-increases. CONCLUSIONS: We concluded that CP and NCP biotypes of BVDV had a significant effect (p ≤0.05) on survival, plasma membrane integrity, and motility of sperm cells in vitro.