Prediabetes (PreDM) and obesity increase the risk of type 2 diabetes. Individuals with these conditions often consume diets higher in animal protein than in plant protein, which are associated with elevated levels of dietary advanced glycation end products (dAGEs). Increased dAGE intake has been linked to blood glucose abnormalities, oxidative stress, and dysbiosis of the microbiota, all of which exacerbate metabolic disorders. Black soybeans, as a plant-based protein source, contain substantially lower levels of dAGEs compared with pork. This study aimed to investigate the effects of substituting animal protein with black soybeans on advanced glycation end product (AGE) levels, oxidative stress, and the gut microbiota in individuals with both PreDM and obesity. This study was a randomized crossover intervention trial conducted over 16 weeks. We recruited men and women aged 20-64 years with both prediabetes and obesity. This study had four periods: 0-4 weeks for the run-in period, 4-8 weeks and 12-16 weeks for the pork or black soymilk intervention period, and 8-12 weeks for the wash-out period. During the intervention period, the participants consumed pork or black soymilk with similar protein content as their dietary protein source. The participants maintained 3 day dietary records, and we measured anthropometric items and collected blood and fecal samples for analysis. The results showed that partially substituting pork with black soymilk as a dietary protein source for 4 weeks significantly reduced dAGE intake. The black soymilk group also exhibited significantly lower blood AGE fluorescence intensity, oxidative stress, and levels of glycative stress markers. Furthermore, black soymilk consumption significantly increased the relative abundance of short-chain fatty acid-producing genera compared with pork consumption. In conclusion, partially substituting dietary pork with black soymilk may reduce serum AGE levels, reduce oxidative and glycation stress, and increase the abundance of short-chain fatty acid-producing microbiota in individuals with both PreDM and obesity. Registration number of Clinical Trial: NCT05290519 (ClinicalTrials.gov).