Accessory sex gland (ASG) in Eriocheir sinensis is the important component of the male reproductive system; its function is still unknown. Here we report that ASG protein can digest the spermatophore wall effectively. By optimizing the incubation temperatures, durations, and secreted protein concentrations using the L₃₀ (5²) orthogonal method, we find the protein concentration was the important factor affecting spermatophore wall digestion. In order to find out the digestion effect and spermatozoa quality, we observed the acrosin activity and survival rate of free spermatozoa obtained by different methods. We found that the survival rate of ASG protein digestion is the highest (98.06%), and its acrosin activity of free spermatozoa was significantly higher (4.290 ± 0.095 µIU/10⁶) than those obtained by traditional trypsinase digestion (2.446 ± 0.251 µIU/10⁶) or mechanical homogenate (1.423 ± 0.109 µIU/10⁶) methods, but it was still significantly lower than those obtained from the spermatheca of mated females (21.729 ± 0.138 µIU/10⁶). Thus, there must be some cooperation between the ASG and other related fertilization organs to improve the acrosin activity of free spermatozoa in vivo. Hence, we colleted the spermatozoa isolated from spermatophores using the trypsinase method and incubated with homogenates from seminal vesicle, accessory sex gland, and spermatheca alone or in combination. We observed that acrosin activity was greatest in samples incubated with an equal mixture of spermatheca and accessory sex gland protein homogenates. We conclude that the accessory sex gland can effectively digest the spermatophore wall to release free spermatozoa and interact with proteins from spermatheca to increase acrosomal enzyme activity of free spermatozoa.