Glucose is suggested to play a key role in motility hyperactivation of mammalian spermatozoa. The current study aimed to investigate the modulatory effects of silymarin and/or a protein kinase A (PKA) inhibitor (H-89) on glucose-mediated motility parameters of mouse spermatozoa. Spermatozoa were incubated in HEPES medium containing normal (NG; 5.5mM) or high (HG; 25mM) glucose concentration. The results of computer-assisted analysis showed that samples incubated in HG resulted in a larger (p<0.05) percentage of motile spermatozoa at 120min (59.5±14.8% vs. 34.0±4.4%) compared to those incubated in NG. The average pathway velocity (VAP), curvilinear velocity (VCL), and straight-line velocity (VSL) exhibited similar patterns at 60 and 120min when incubated in HG (p<0.05). Treatments with silymarin (5, 10, 20μg/mL) did not significantly affect sperm motility under NG conditions, but decreased the HG-enhanced motility, VAP, and VCL at 120min (p<0.05). H-89 (30μM) reduced (p<0.05) motility at 30min examined in the NG or HG medium. At 90min, H-89 also reduced (p<0.05) the HG-enhanced motility of spermatozoa incubated with or without 20μg/mL silymarin by 49% or 32%, respectively. In conclusion, the H-89-inhibition of glucose-mediated mouse sperm motility and certain types of velocity suggests that the glycolysis–PKA pathway is involved in the regulation of sperm motility. Silymarin may maintain sperm motility under NG conditions, but it inhibits glucose-activated sperm motility.