Abstract: In order to explore the regulation rule of spermatozoa motility of Schizothorax irregularis, and to improve its fertilization rate and fingerling production efficiency, we applied a significance test and response surface method to study the single regulation and compound regulation of glucose, glycerol and three salts on sperm motility of S. irregularis. The results show that the maximum fast movement time of S. irregularis spermatozoa was 37.00 s when glucose was 433.7 kPa, significantly higher than those in the other groups (P<0.05). The maximum fast movement time of S. irregularis spermatozoa was 32.33 s when glycerol was 506.7 kPa, without difference with that in 416.3 kPa group, but was significantly higher than those in the other groups (P<0.05). The maximum fast movement time of S. irregularis spermatozoa was 35.00 s when sodium chloride was 431.4 kPa, without difference with that in 348.8 kPa group, but was significantly higher than those in the other groups (P<0.05). Potassium chloride had no obvious effect on activating spermatozoa, and magnesium chloride solution showed inhibition effect for spermatozoa. An equation about the relation of fast movement time and glucose, glycerol and sodium chloride was established. The linear and quadratic effects of the equation were very significant (P<0.01), and interaction effect was significant (P<0.05). The optimizied formulation of model was sodium chloride of 164.78 kPa, glycerol of 216.55 kPa, glucose of 208.43 kPa, and the theoretical FT was 68.24 s. The results indicate that the optimized activator formula by response surface methodology can improve the motility of S. irregularis spermatozoa.