Objective: The recent advent of flow cytometry (FCM), coupled with fluorescent dyes, has been success-fully applied to assess mitochondrial function. The aim of this study was to investigate the feasibility and clinical significance of detecting sperm mitochondrial function and to evaluate sperm mitochondrial function by using Rhodamine 123/propidium (Rh123/PI) dual fluorescent staining and FCM in asthenospermia and oligoastheno-zoospermia. Methods: Twenty-five fertile men (with normal sperm parameters) and 230 infertile patients were examined. Fifty-five patients of the above 230 patients were selected for idiopathic infertility samples and were divided into two groups: asthenospermia (n = 30) and oligoasthenozoospermia (n = 25). Rh123/PI dual fluorescent staining and FCM were carried out to examine sperm mitochondrial function. Results: Significant differences were found between the normal and abnormal semen samples (P < 0.05) when Rh123+/PI-, Rh123-/PI+ and Rh123-/PI- sperm were examined by FCM, but there was no significant difference between the asthenospermia (P = 0.469) and oligoasthenozoospermia group (P = 0.950) when Rh123+/PI- and Rh123-/PI+ sperm were then examined; how-ever, a significant difference was found between the 2 groups (P = 0.003) when Rh123-/PI- sperm were examined. There was no correlation between Rh123-/PI- sperm and semen parameters in the normal group, but there was a significant negative correlation between the sperm concentration and Rh123-/PI- sperm in asthenospermia and oligoasthenozoospermia patients (r = -0.509, -0.660; P = 0.018, 0.038). Conclusion: Rh123/PI dual fluorescent staining and FCM can provide reliable information to assess the quality of sperm and reveal differences in mito-chondrial membrane potential in asthenospermia and oligoasthenozoospermia.