Anthocyanin, as a typical food bioactive molecule, is capable of reversing inflammatory, oxidative and allergic condition thus contributes to intestinal health. We were wondering whether anthocyanin has influence on the infiltration of inflammatory cells into the intestinal mucosa and thus help enhancing intestinal barrier which could be damaged in some metabolic diseases. In this study, the influence of anthocyanin (administered orally) on the alterations (including structure and permeability) of the intestinal mucosa in mice in response to a high fat-high cholesterol (HFHC) diet was investigated. Primary T helper 17 (Th17) cells were isolated from mouse intestine tissues to observe the modulatory role of anthocyanin through the transcription phosphorylated STAT3 (p-STAT3). The results indicated that anthocyanin significantly alleviated HFHC-induced impairment in the intestinal structures and permeability in a dose-dependent manner; moreover, anthocyanin appeared to inhibit HFHC induced the expression of p-STAT3, thereby disturbing Th17 cell differentiation. In high-fat diet (HFD, cholesterol level non-modified)-challenged mice selective p-STAT3 inhibitor significantly reversed the effects of anthocyanin, which were decreased amount of interleukin (IL)-17A (produced and released from Th17 cells) and the protected intestinal structure/function. In summary, the results of this study suggest that anthocyanin may attenuate the damage of intestinal barrier in HFHC mice through regulating intestinal STAT3-Th17-IL-17A signal transduction pathway.