Objective To investigate the influence of culture temperature and concentration of fluorescence dye Hoechst 33258 (H258) on vital labeling of human sperm. Methods Fourteen semen samples were randomly selected, and were cultured with 0 mg/mL (control), 0.01 mg/mL and 0.1 mg/mL H258 for 1 h under different temperatures (0 ℃, room temperature and 37 ℃) after washing. Progressive motility of human sperm was assessed, and H258 staining rates of the sperm were recorded. Semen samples without treatment were served as blank controls. Results Under the conditions of this research, H258 concentrations and culture temperatures had significant impact on sperm motility. The percents of progressive motility of sperm gradually decreased with the increase of H258 concentrations and decrease of culture temperatures (P<0.01). The staining rates of the sperm gradually increased with the culture temperatures and mass concentrations of H258, and there were significant differences among staining rates of the sperm cultured with different temperatures at the mass concentration of 0.1 mg/mL (P<0.01). Conclusion H258 can be used in vital labeling of human sperm. The optimal staining condition was 37 ℃+0.1 mg/mL H258 in this study. Culture condition should be determined by specific purpose.