Direct conjugation of histidine to poly(2-hydroxyethyl aspartamide) (PHEA-His) and C18-grafted PHEA (PHEA-g-C18-His) was achieved via an ester linkage using N(alpha)-Boc-L-histidine, followed by the deprotection of Boc groups. PHEA-His series would be expected as an endosomolytic synthetic polymer because of the buffering capacity at physiological and endosomal pH regulated by alpha-amine and imidazole groups in side chains. PHEA-g-C18-His series formed stable self-aggregates due to the hydrophobic interaction between grafted alkyl chains. The size, zeta potential, and micropolarity of PHEA-g-C18-His series greatly increased at pH 5.0, because aggregates swelled by a positive surface charge and the electrostatic repulsion of ionized histidine moieties in the aggregate surface. In the confocal microscopy, it was revealed that PHEA-g-C18-His was more uniformly distributed than PHEA-g-C18 in HeLa cells after 8 h of incubation and was attributed to the endosomolytic ability of conjugated histidine moieties. In doxorubicin-loaded self-aggregate systems, the histidine conjugation improved the cell cytotoxicity by a fast release of loaded doxorubicin at low pH and the endosomolytic ability of conjugated histidine, resulting in the easy nuclear access of doxorubicin.