Objective To explore a method for the isolation and extraction of murine gut muscu-laris cells.Methods A"descending ladder"dissociation solution was used to dissociate the mucus,mu-cosa,lamina propria and submucosa.The concentration of trypsin in the mucus,mucosal and submucosal dissociation solutions gradually decreased.The digestion solution for muscularis consisted collagenaseⅠ/Ⅳ,neutral protease and Deoxyribonuclease Ⅰ(DNase Ⅰ).The trypsin concentration(wt%)gradients of experimental groups 1,2 and 3 were as follows:0.50,0.25,0.10;0.50,0.30,0.20;1.00,0.50,0.25.T-test was used to compare between the groups.Results The muscularis was successfully exposed and the muscularis cells were obtained.The exposure proportion of muscularis was(91.00±1.87)%,and the cell viability was(79.20±0.49)%.The exposure ratio of muscularis in experimental group 1 was sig-nificantly higher than that in experimental group 2[(91.00±1.87)%vs.(80.00±3.16)%,t=2.994,P<0.05]and group 3[(91.00±1.87)%vs.(83.00±2.55)%,t=2.530,P<0.05].The cell activi-ty in experimental group 1 was also significantly higher than that in experimental group 2[(79.20± 0.49)%vs.(70.20±0.37)%,t=14.600,P<0.01]and group 3[(79.20±0.49)%vs.(62.40± 0.40)%,t=26.560,P<0.01].Conclusion This new method could isolate murine muscularis cells with high viability.