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As an important medicinal intermediate with broad uses, validamine, an aminocyclitol, isolated from the enzymolysis broth of validamycins, has gained more and more attention. The absolute configuration of validamine is similar to that of α-D-glucose, and it demonstrates powerful inhibition activity on glycosidase. In this paper, the inhibitory effect of validamine on porcine small intestinal sucrase was investigated. Validamine was found to be a Validamine exhibited a dose-dependent inhibition effect on porcine small intestinal sucrase, whereby the inhibition interaction of validamine and porcine small intestinal sucrase was a fast binding process. The inhibition of validamine on porcine small intestinal sucrase was pH-dependent.