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Objective To evaluate effect of recombined human tumor necrosis factor (rhTNF-α)on mitochondrial transmembrane potential and motility of human sperm in vitro Methods Semen samples for study were obtained from 40 health men (average age 26± 1.2 years) with normal semen analysis. Sperm suspension with computer aided of semen analysis (CASA) technique; 2) were stained in the presence of 10 μg/ml Rh123and PI, mitochondrial transmembrane potential of those was analyzed by flow cytometry(FCM).Results Significant differences were found between experimental groups and control groups on viability, straight line velocity, curvilinear velocity, average path velocity,progressive motility of human sperm and number of sperm with normal mitochondrial transmembrane potential (P＜0. 01) expect final concentration 30 pg/ml group (P＞0.05). Sperm motility lowed with increasing rhTNF-α concentration and incubating time (P＜0.01). Number of sperm with normal mitochondrial transmembrane potential decreased with increasing rh TNF-a concentration and incubating time (P＜0.01).Conclusion rhTNF-α can decrease human sperm motility function in vitro, which can interfere the function of human sperm mitochondrial transmembrane potential and may inhibit sperm mitochondrial enzymatic activities.