This study aimed to investigate the effects of Sargassum Crude Polysaccharide (III) complex (ASP-Cr (III)) on glucose metabolism and gut microbiota regulation in diabetic mice. Spontaneous type 2 diabetic db/db mice were selected as the experimental model and divided into seven groups: normal control group (NC), model group (DC), metformin group (MET), Sargassum Crude Polysaccharide group (ASP), inorganic chromium group, low-dose ASP-Cr (III) group, and high-dose ASP-Cr (III) group. All groups received intragastric intervention for 4 weeks. The acute toxicity test showed that ASP-Cr(III) at a dose of 1500 mg·kg-1·d-1 had no acute toxicity in mice. animal experiments indicated that mice in the model group remained in a persistent hyperglycemia state, with a blood glucose level of 26.78 mmol·L-1 after 4 weeks. Both low-dose and high-dose ASP-Cr (III) interventions significantly reduced fasting blood glucose (FBG) to 17.18 mmol·L-1 and 19.6 mmol·L-1, respectively (P<0.05). Compared with the DC group, ASP-Cr (III) treatment increased the renal index by 73.19% and 45.16%, decreased the hepatic index by 26.34% and 17.81%, and elevated the pancreatic index by 51.33% and 36.82%, along with alleviated pathological lesions of liver tissue.western blot (WB) analysis demonstrated that ASP-Cr (III) significantly upregulated the protein expression of AKT2 and downregulated the protein expression of PI3K and PCK1(P<0.05), with a therapeutic effect superior to that of the ASP group and inorganic chromium group. Intestinal microbiota analysis revealed that ASP-Cr (III) significantly enhanced the richness and diversity of gut microbiota (P<0.05), regulated the Firmicutes/Bacteroidetes ratio, and ameliorated the structural imbalance of intestinal microbiota. In conclusion, ASP-Cr(III) can ameliorate glucose metabolism disorder by repairing liver tissue, regulating the expression of insulin signaling-related proteins, and modulating gut microbiota. This study provides experimental evidence for the development of ASP-Cr (III) as a novel anti-diabetic functional factor or chromium supplement.