Simple SummarySea cucumbers are very sensitive to the influence of environmental factors, and will vomit their intestines and undergo tissue degradation under certain stimuli. In order to determine the effect of endogenous enzymes in the sea cucumber intestine on the body wall during body wall softening, intestine extracts and crude collagen fibers were prepared, respectively. The endogenous enzymes belonging to serine proteases were identified by gelatin zymography. The rheological property results showed that the addition of sea cucumber intestinal extracts reduced the viscosity of crude collagen fibers from 32.7 Pa·s to 5.3 Pa·s. The serine protease inhibitor phenylmethylsulfonyl fluoride inhibited the activity of intestinal extracts, and maintained the viscoelasticity of crude collagen fibers. In this paper, endogenous enzymes in sea cucumber intestinal extracts were identified as serine endopeptidases, which caused softening of the body wall.AbstractSea cucumbersApostichopus japonicuswill vomit their intestines during certain stimulations, and the collagen of the body wall will then be degraded. To define the effect of the sea cucumber intestine extracts on the body wall, the intestinal extracts and crude collagen fibers (CCF) of sea cucumberA. japonicuswere prepared. According to the gelatin zymography, the type of endogenous enzymes in intestinal extracts were mainly serine endopeptidases with optimal activities at pH 9.0 and 40 °C. According to the rheology results, the viscosity of 3% CCF decreased from 32.7 Pa·s to 5.3 Pa·s by adding intestine extracts. The serine protease inhibitor phenylmethanesulfonyl fluoride inhibited the activity of intestinal extracts and increased the viscosity of collagen fibers to 25.7 Pa·s. The results proved that serine protease in the intestinal extracts participated in the process of body wall softening in sea cucumbers.