Plant-based proteins have received considerable global attention due to their nutritional value and potential health effects. As a high-quality plant protein, the hypoglycemic effect of quinoa protein and its potential mechanism have not been fully elucidated. In the present study, we compared the hypoglycemic effects of raw quinoa protein (RP-quinoa) and heat-treated quinoa protein (HP-quinoa) and further explored their potential mechanisms using multi-omics analysis based on gut microbiota and fecal metabolic profiles in HFD-fed mice. Our results showed that both RP-quinoa and HP-quinoa effectively improved glucose metabolism and protected against alterations in gut microbiota induced by a chronic HFD. In addition, quinoa protein increased the relative abundance of beneficial bacteria such as the g__Lachnospiraceae_NK4A136_group , g__Eubacterium_xylanophilum_group , and g__Negativibacillus , followed by an increase in short-chain fatty acids and potentially beneficial metabolites such as L-phenylalanine and L-cysteine. Together, these findings provided the basis for linking gut microbiota and their metabolites to the hypoglycemic effect of quinoa protein.