Postprandial hyperglycemia is an early indication of type 2 diabetes and the target of many anti-diabetic and anti-obesity studies. α-Glucosidase and α-amylase are the crucial factors in regulating starch digestion and glucose absorption, making them key targets for many studies to treat postprandial hyperglycemia. We studied the inhibitory activities of microalgal fucoxanthin against rat-intestinal α-glucosidase and pancreatic α-amylase along with the antidiabetic effect to induce differentiation in 3T3-L1 pre-adipocytes using Oil Red-O staining. Fucoxanthin displayed strong hindrance activities toward α-amylase in a concentration-dependent manner, with an IC₅₀ value of 0.68 mmol/L, whereas weak inhibitory activity against α-glucosidase, with an IC₅₀ value of 4.75 mmol/L. Fucoxanthin also considerably elevated glucose oxidase activity in 3T3-L1 cells by 31.3% at 5 µmol/L. During adipocyte differentiation, fucoxanthin showed lipid accumulation in 3T3-L1 cells with no cytotoxicity up to 20 µmol/L. However, fucoxanthin had no inhibitory activity on glucose-6-phosphate dehydrogenase. These results suggest that fucoxanthin might be useful for the prevention of obesity or diabetes by inhibiting carbohydrate-hydrolyzing enzymes and lipid accumulation and be utilized as an ingredient for a functional food or dietary supplement.