The aryl hydrocarbon receptor (AhR) pathway is now considered an important pharmacological target for many human diseases. This study examined the effects of grape seed extract (GSE) obtained from the Koshu grape variety on AhR activity. The Koshu-derived GSE inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated AhR activation in vivo and in vitro. These results suggest that Koshu GSE has an antagonistic effect on TCDD-induced AhR activation. Furthermore, gavage administration of TCDD resulted in the elevation of liver function, but administration of Koshu GSE together with TCDD did not elevate serum aspartate aminotransaminase (AST) and alanine transaminase (ALT) levels. These results suggest that Koshu GSE may have the potential to prevent liver disorders.