Fish protein is not only nutritional but also promotes health by improving insulin sensitivity and hypercholesterolemia. Few studies have examined the relationship between gut microbiota and the enhanced insulin sensitivity due to the intake of Alaska pollock protein (APP). Hence, we assessed the glycolytic enzyme inhibitory activity of APP in in vitro study and the alteration of blood glucose level in insulin tolerance test (ITT) and glucose tolerance test (GTT) and gut microbiota following APP intake in the in vivo study. In initial experiments, the glycolytic enzyme (α-amylase, α-glucosidase, and sucrase) inhibitory activities of APP and its digest were not drastically altered compared with that of casein and its digests. In further experiments, rats fed an AIN-93G diet containing 20% (w/w) casein or APP for 8 weeks, and the composition of fecal microbiota analyzed by 16S rRNA amplicon sequence analysis. In addition, at 6 and 7 weeks of administration of experimental diet, insulin and glucose tolerance tests were evaluated, respectively. Compared with dietary casein, dietary APP has blood glucose-lowering activity as evident in the ITT and GTT. Moreover, APP group altered the structure of fecal microbiota, and area under the curves of the ITT and GTT and the relative abundance of Blautia, which is associated with glucose metabolism, tended to be positively correlated (P = 0.08 and 0.10, respectively). This study illustrates a novel finding that APP intake could alter the composition of gut microbiota and improve insulin sensitivity.