The purpose of this article was to better understand the role of lignin in the recalcitrance of lignocellulosic biomass during enzymatic hydrolysis. Steam exploded wheat straw was partially delignified with sodium chlorite to six different grades of delignification. Delignification did not have a significant impact on the enzymatic hydrolysis of the studied wheat straw in the experimental conditions tested. Inhibitive impact of lignin in terms of non-productive adsorption was then explored using soda lignin from wheat straw and kraft lignin from softwood. The addition of both lignins had a strong negative influence on the hydrolysis of highly crystalline cellulose (Avicel), whereas it impacted only slightly the hydrolysis of delignified wheat straw. These results are probably linked to the greater accessibility and surface area of steam exploded wheat straw cellulose, which are much higher than those of the crystalline cellulose Avicel.