Abstract: This experiment was conducted to investigate the variation of digestive enzyme activities in the simulated gastric and small intestinal digestion for pigs, which will provide a reference to develop the simulated digestion method for determining nutrients digestibility for pigs. In experiment 1, a single factor completely randomized design was used to investigate the variation of digestive enzyme activities depending on digestion time when the simulated gastric and small intestinal fluid were injected once during the simulated digestion for corn, soybean meal, wheat bran and corn-soybean meal. The digestion time of 0, 1, 2, 3 and 4 h was arranged for gastric digestion phase. The specific and total activities of pepsin in the digestive fluid were determined. The digestion time of 0, 2, 4, 6, 8, 12 and 16 h was arranged for the small intestine digestion phase. The specific and total activities of trypsin, chymotrypsin and amylase in the digestive fluid were determined. In experiment 2, the digestive enzymes were supplemented at 4 h of small intestine digestion phase according to the results of experiment 1. The digestive enzyme activities were determined at 4, 6 and 8 h of small intestine digestion phase after supplementing digestive enzymes. Each in vitro digestion time was considered as a treatment, and each treatment contained 5 replicates with 1 digestion tube per replicate. The results showed that:1) When the simulated gastric fluid was mixed with corn, soybean meal, wheat bran or corn-soybean meal, the specific activity of pepsin in the digestive fluid was rapidly reduced (accounting for 28.8% to 59.1% of initial activity). However, from 1 to 4 h of the simulated digestion, the specific and total activities of pepsin were increased with the quadratic curve(P<0.05). The final values were 78.0% to 96.1% relative to initial specific activity and 87.2% to 102.7% relative to initial total activity of simulated gastric fluid, respectively. In the small intestine digestion phase, different extent of reduction of activities of amylase, trypsin and chymotrypsin in simulated digestive fluid was observed (P<0.05). The quadratic or non-linear curve variation of amylase and chymotrypsin activity in digestive fluid was observed with different substrates(P<0.05). The reduction of trypsin activity in digestive fluid with quadratic curve was observed (P<0.05). The greatest reduction of activity was observed in trypsin. 2) After the digestive enzymes were supplemented in simulated small intestinal digestion, the specific activities of amylase, trypsin and chymotrypsin in the digestive fluid could be recovered to the initial value, but they still decreased with the digestion time(P<0.05), among them, the trypsin activity still decreased rapidly. In conclusion, in the process of simulated digestion, there is no need to supplement pepsin in the gastric digestion stage; the digestive enzyme needs to be supplemented in the small intestine digestion stage.