Objective To observe the changes to, and possible mechanism of, intestinal permeability in pigs without direct injury after an abdominal- and intestinal-penetrating injury from firearms in cold environment at high altitudes. Methods Fifty-five experimental pigs were divided into two groups: high-altitude cold group (HC) and low altitude normal temperature group (LN). According to the observation time, each group was divided into five experimental subgroups: 0 h, 2 h, 4 h, 8 h, and 24 h. There were six pigs in each HC subgroup and five pigs in each LN subgroup. After euthanasia, intestinal tissues were taken, and the levels of the inflammatory factors TNF-α, and IL-6 in intestinal homogenate and the concentrations of intestinal permeability-related proteins DAO and D-lactate acid in blood were detected by ELISA method. The intestinal tissues of experimental pigs were taken at 0 h and 8 h for LN and 8h for HC, and intestinal pathological changes were observed and scored after HE staining. The concentrations of Occludin, ZO-1,Claudin-3, TLR4, NF-κB, and MLCK (proteins related to intestinal permeability) were detected by Western blot to explore the effect of a cold environment at high altitude on secondary intestinal permeability changes after injury and the possible mechanisms. Results Both the HC group and LN group experienced typical abdominal intestinal penetrating injuries, and there were no significant differences in their abdominal infection scores or intestinal adhesion (P>0.05). The levels of DAO and D-LA in the serum of experimental pigs in the HC and LN groups gradually increased over time. The levels of DAO and D-LA in the HC group were significantly higher than those in the LN group at all time points (P<0.01 or P<0.001). The fastest increase in DAO and D-LA in the HC group was 4 h to 8 h, while in the LN group, it was 8 h to 24 h. The pathological score of intestinal tissue in the HC group was significantly higher than that in the LN group of experimental pigs (P<0.01). The inflammatory factors TNF-α and IL-6 both increased over time in the intestinal tissue of LN and HC groups. The most significant time point for a increase of inflammatory factors in the HC group was 4 h to 8 h, while in the LN group, it was 8 h to 24 h. The intestinal tissue IL-6 and TNF-α levels of experimental pigs in the HC group were higher than those in the LN group the entire time (P<0.05, P<0.01, or P<0.001). The levels of occludin and ZO 1 in the HC group at 8 h decreased significantly compared to those of the LN group at the 8 h time point (P<0.05), while claudin-3 showed a significant decrease in LN (P<0.001). In the HC group, TLR4, NF-κB, and MLCK were both higher than those in the LN group at 8 h, and the difference was statistically significant (P<0.05). Conclusions A high altitude cold environment can lead to a secondary increase in intestinal permeability after abdominal-penetrating firearm injury, and its mechanism may be related to the TLR4/NF-κB/MLCK pathway.