To evaluate the role of flow cytometry in semen assessment.Semen samples from 104 infertile male patients (as the case group) and 10 fertilized donors (as the control group) were analyzed for the volume of ejaculate and sperm concentration, motility and atypical morphology by computer-assisted semen analysis (CASA), the viability, chromatin structure and mitochondrial membrane potential (MMP) of the sperm stained by SYBR-14/PI, AO and JC-1 respectively, and assessed with flow cytometry. The results were analyzed through SAS software.U tests indicated that the semen from the infertile patients had not only lower concentration (U = 2.51, P = 0.0143), lower motility (U = 3.44, P = 0.001) and higher rate of atypical morphology (U = -5.88, P < 0.0001), but also lower viability (U = 4.72, P < 0.0001), MMP (U = -2.53, P = 0.0309), and chromatin integrity (alpha t: U = -3.82, P = 0.0003; SD alpha t: U = -3.98, P = 0.0001; COMP alpha t: U = -3.57, P = 0.0005). The multiple stepwise regression analysis revealed that sperm motility was positively correlated with sperm membrane integrity (t = 1.66, P = 0.1016), sperm MMP (t = 3.33, P = 0.0014) and sperm acrosome integrity (t = 3.24, P = 0.0019), while sperm MMP was negatively correlated with the rates of sperm neck and tail defects (t = -3.44, P = 0.001).Flow cytometry plays a significant role in the evaluation of the quality of human sperm, and can be adopted as a useful tool in the diagnosis and treatment of male infertility.