Spiny burs of Castanea mollissima Blume (Chinese chestnut) are usually discarded as industrial waste during post-harvesting processing. The objective of this study was to establish an extraction and isolation procedure for tannins from chestnut burs, and to assess their potential antioxidant activity. Aqueous ethanol solution was used as extraction solvent, and HPD 100 macroporous resin column was applied for isolation. The influence of solvent concentration in the extraction and elution process on extraction yield, tannins and polyphenols content, as well as antioxidant potential, including DPPH and ABTS radical scavenging ability, reducing power ability and cellular antioxidant ability were assessed. In both the extraction and isolation process, 50% aqueous ethanol led to superior total tannins and polyphenols content as well as significantly higher antioxidant activity. In addition, the antioxidant activity and the total tannins content in extracts and fractions had a positive linear correlation, and the predominant components responsible for antioxidant activities were characterized as hydrolysable tannins. To the best of our knowledge, this is the first report on the enrichment of tannins from burs of C. mollissim using macroporous resin chromatography, and to assess the cellular antioxidant activity of them.