The study aimed to explore the digestion of a myoglobin diet and its relationship with the gut microbiota and intestinal barrier at two feeding time points. The study indicated that myoglobin diets had a higher α-helix content and lower digestibility. The particle sizes of the digested myoglobin diets were higher but their zeta potential values were lower than those of a casein diet. C57BL/6J mice were fed with a casein diet incorporated with 0, 0.38%, 1.13% and 3.39% myoglobin for 3 and 8 weeks. With the increase of the myoglobin content, the relative abundance of increased significantly. Moreover, the content of short chain fatty acids tended to increase and then decrease as the myoglobin content increased at 3 weeks. Furthermore, the low-myoglobin diet upregulated the gene expression involved in colonic mucin and tight junction proteins by increasing the proportion of beneficial microbiota. However, the high-myoglobin diet had adverse effects.