The inhibition or delay of starch digestion by dietary compounds could help manage postprandial blood glucose levels. The objective of this study was to identify constituents from whole grain blue wheat capable of decreasing α-amylase-catalyzed starch digestion. An activity-guided fractionation approach based on liquid chromatography was used to identify solvent-and alkaline-extractable blue wheat constituents reducing α-amylase-mediated starch digestion in vitro. Fatty acids, potentially released from cell wall polymers by alkaline hydrolysis, inhibited the digestion of amylose, probably through the formation of amylose-lipid complexes. However, the degradation of amylopectin was not affected by fatty acids. In addition, 1-(3,5- dihydroxyphenyl)heneicosan-2-one, a 5-(2'-oxoalkyl) resorcinol, was found to reduce starch digestion. However, because the digestion of both amylopectin and amylose was reduced, the inhibition mechanism was different from that of fatty acids. Further research is needed to evaluate whether this component also reduces starch digestion in vivo. Other phenolic compounds of blue wheat such as anthocyanins or hydroxycinnamates were not identified as major starch digestion inhibitors by using the activity-guided fractionation approach.