Antigen presentation on major histocompatibility complex (MHC) class I and subsequent priming of antigen-specific cytotoxic T lymphocytes (CTLs) are essential steps for vaccination but exogenous soluble proteins are conventionally taken up by endosomes and presented on MHC class II rather than class I. In this study, we demonstrated, for the first time, that ovalbumin (OVA) chemically cationized with hexamethylenediamine (HMD) can induce OVA-specific CTLs without any adjuvants. Cationization of OVA greatly enhances cellular uptake by antigen-presenting cells (APCs) through adsorptive endocytosis. Two kinds of Cat-OVAs with different cationic charges were evaluated to elicit a CTL response through enhanced uptake by APCs and concomitant participation in the class I pathway. Cat₂₀-OVA, a cationized OVA derivative with more cationic charges, showed pronounced induction of the OVA-specific CTL response after subcutaneous immunization. The CTL response was comparable with that induced by OVA with CFA. In contrast to the CFA formulation that actually produced local tissue damage in this study, local damage at the injection sites was not observed with Cat-OVAs. Cat₂₀-OVA also showed a significant protective effect on the growth of OVA-expressing E.G7 tumor cells. In conclusion, cationization of soluble antigen is a useful and safe vaccination strategy.