Consuming low-glycemic index foods, such as black highland barley (BHB), is essential for regulating post-meal glucose levels in individuals with type 2 diabetes mellitus (T2DM). This study examined how the starch structure of BHB affected glucose fluctuations. The incorporation of BHB enlarged starch granule size and modified the crystalline structure to an A + Ⅴ type with a relative crystallinity of 31.14%. This alteration facilitated a more organized release of glucose. Population-based trials lasting 6 weeks had demonstrated that that BHB could reduce glucose variation, ghrelin levels, and insulin resistance in T2DM. Additionally, it increased the populations of beneficial gut bacteria such as Akkermansia, Bifidobacterium, and Lactobacillus, while also raising levels of gastric inhibitory peptides, phenolics, and specific gut metabolites. The specific digestion protocol and mathematical model of BHB disclosed its slow gastric emptying and glucose release rates, which were crucial for maintaining stable glucose levels in individuals with T2DM. Understanding the starch structure of BHB provided an effective dietary intervention for managing T2DM-related glucose fluctuations.