AbstractThis study aimed to investigate the effects and underlying mechanisms of real meat and plant‐based meat analogs on intestinal barrier function. Methods including PacBio sequencing, HPLC, histological observation, western blotting, and RT‐qPCR were jointly applied. The results indicated that the 20‐day intake of real meat and plant‐based meat analogs did not induce pathological changes in the colon of mice. However, when the feeding period was extended to 68 days, inflammatory infiltration was observed in the colon tissue of mice fed plant‐based meat analogs. In contrast, the intake of real meat for 68 days performed better in maintaining colonic barrier function by reducing cell apoptosis, increasing the number of goblet cells and mucin secretion, and upregulating the expression of tight junction proteins. High‐throughput sequencing revealed that the intake of real meat significantly altered the composition of the gut microbiota, reducing pro‐inflammatory bacteria (such as Bacteroides, Desulfovibrio, or Colidextribacter) or pathogenic bacteria (such as Mucispirillum and Helicobacter) while increasing beneficial bacteria (such as Lactobacillus, Bifidobacterium, or Roseburia). Additionally, the consumption of real meat markedly decreased protein fermentation in the colon, leading to lower levels of cadaverine, putrescine, and spermine. These changes associated with real meat consumption reduced the activation of the pro‐inflammatory pathway (lipopolysaccharide/Toll‐like receptor 4/nuclear factor kappa B), ultimately maintaining colonic homeostasis in mice. Collectively, the findings suggest that the proper intake of real meat is more beneficial for colon health in mice compared to plant‐based meat analogs.