In this study, five types of mono-acylated anthocyanins were isolated from colored-fleshed potato (Solanum tuberosum L.) and black carrot (Daucus carota subsp. sativus), and their gastrointestinal absorption was evaluated based on the plasma profiles of rats. The absorption amounts of mono-acylated anthocyanins were slightly higher or at least similar to those of non-acylated anthocyanins, which carry the same aglycone, when the purified anthocyanins were orally administered. The absorption amounts of petunidin 3-O-(6′′-O-(4′′′-O-p-coumaroyl-α-L-rhamnopyranosyl)-β-D-glucopyranoside)-5-O-β-D-glucopyranoside isolated from purple-fleshed potato were 1.3 to 2.3-fold higher than those of three types of cyanidin 3-O-(2′′-xylopyranosyl-6′′-O-(6′′′-O-acyl-β-D-glucopyranosyl))- β-D-galactopyranosides isolated from black carrot, whereas pelargonidin 3-O-(6′′-O-(4′′′-O-p-coumaroyl-α-L-rhamnopyranosyl)-β-D-glucopyranoside)-5-O-β-D-glucopyranoside isolated from red-fleshed potato exhibited an exceptionally poor absorption profile, as demonstrated by the comparison of the area under the plasma concentration curves during 8 h after oral administration normalized to the orally administered dose. The gastrointestinal absorption of mono-acylated anthocyanins was tended to determine by their structures, including aglycone moieties, attached sugars, and the balance of the entire molecule. Our findings suggested that the branched acylated sugars attached to position 3 tended to suppress the absorption of mono-acylated anthocyanins from the gastrointestinal tract. Furthermore, the poor intestinal absorption of mono-acylated anthocyanins in the mixtures was likely due to the competitive absorption of these molecules with contaminants that co-occur in plant materials such as organic acids.