Black soybean contains various polyphenols such as flavan-3-ols and anthocyanins in seed coat and isoflavones in cotyledon. The content of polyphenols and their functions may vary depending on the variety and cultivation year. In this study, we measured the content of polyphenols in two black soybean varieties, Tanbakuro and Kurotsuru, over five cultivation years and investigated their antioxidant activity. Total polyphenol content in Tanbakuro was lower than that in Kurotsuru. Polyphenol contents in seed coat and cotyledon showed reverse trends during cultivation years. Variations in the polyphenol content and antioxidant activity in the seed coat and cotyledon showed the same trend. We focused on flavan-3-ols and expanded experiments using Caco-2 and HepG2 cells. (-)-Epicatechin and procyanidin B2 were easily permeated through Caco-2 cells than procyanidin C1 and cinnamtannin A2. Contents of (-)-epicatechin and procyanidin B2 in the permeated solution strongly correlated with their antioxidant activity in HepG2 cells.