β-CATENIN is an evolutionarily conserved multifunctional molecule that maintains cell adhesion as a cell junction protein to safeguard the integrity of the mammalian blood-testes barrier, and also regulates cell proliferation and apoptosis as a key signaling molecule in the WNT/β-CATENIN signaling pathway. In the crustacean Eriocheir sinensis, Es-β-CATENIN has been shown to be involved in spermatogenesis, but the testes of E. sinensis have large and well-defined structural differences from those of mammals, and the impact of Es-β-CATENIN in them is still unknown. In the present study, we found that Es-β-CATENIN, Es-α-CATENIN and Es-ZO-1 interact differently in the testes of the crab compared to mammals. In addition, defective Es-β-CATENIN resulted in increased Es-α-CATENIN protein expression levels, distorted and deformed F-ACTIN, and disturbed localization of Es-α-CATENIN and Es-ZO-1, leading to loss of hemolymph-testes barrier integrity and impaired sperm release. In addition to this, we also performed the first molecular cloning and bioinformatics analysis of Es-AXIN in the WNT/β-CATENIN pathway to exclude the effect of the WNT/β-CATENIN pathway on the cytoskeleton. In conclusion, Es-β-CATENIN participates in maintaining the hemolymph-testes barrier in the spermatogenesis of E. sinensis. •We performed the first molecular cloning and bioinformatics analysis of Es-AXIN.•Es-β-CATENIN has an important role in maintaining the integrity of the HTB.•Defective Es-β-CATENIN leads to increased Es-α-CATENIN protein expression levels.•Es-α/β-CATENIN and ZO-1 interact differently in crab testes compared to mammals.