The digital analysis technique of sperm quality assessment in fish has provided reliable reference evidences for the effects and the damage mechanism of cryopreservation, genetics and breeding, etc. From the study using fish color sperm image analysis systems (FSQAS－2000) conducted on the cryopreserved sperm of Chinese sturgeon (Acipenser sinensis), Amur Sturgeon (A. schrenckii), Siberian Sturgeon (A. baerii) and Paddlefish (Polyodon spathula) under different activating mediums, the most parameters, including the curvilinear velocity (VCL), the straight line velocity (VSL), the angular path velocity (VAP), angular displacement (MAD) and percentage of motile sperm, etc. were compared for the first time in china. And the influences which different activating medium consisted of different osmolality, pH and concentration of Mg2+ made on the parameters were also analyzed in the study. Our results indicated that: First, the osmolality of activating medium for the sturgeons mentioned above was quite low, among them, the lowest for Siberian Sturgeon (10mOsm.kg-1), the highest for Paddlefish (30mOsm.kg-1), the middle for Chinese sturgeon and Amur Sturgeon (20mOsm.kg-1). Second, it had a significant (P≤0.05 or P≤0.01）influence on VCL, VSL, VAP and percentage of motile sperm in cryopreserved samples. Third, pH of activating medium had the most significant difference on MAD (P≤0.01) among the parameters, and it was lower and MAD was lager. Fourth, the most appropriate pH was 7.5 for Siberian Sturgeon and 8.5 for Chinese sturgeon, Amur Sturgeon and Paddlefish. And last, the velocities (VCL, VSL, VAP) had significant increases (P≤0.01), when MgCl2 (5 mmol.L-1) was added into activating medium. In conclusion, the appropriate activating mediums for the cryopreserved sperm of Siberian Sturgeon, Chinese sturgeon and Amur Sturgeon, Paddlefish were 10 mmol.L-1 Tris-HCl＋5 mmol.L-1 MgCl2, pH7.5, 20 mmol.L-1 Tris-HCl＋5 mmol.L-1 MgCl2, pH8.5, 30 mmol.L-1 Tris-HCl＋5 mmol.L-1 MgCl2, pH8.5, respectively. Using the mediums to activate sperm, there were highly positive linear correlations（P＜0.01）in percentage of motile sperm between prefreeze and post-thaw sperm, which R2 was 0.83, 0.99, 0.96, 0.95 for Siberian Sturgeon, Chinese sturgeon, Amur Sturgeon and Paddlefish, respectively.