Previous studies have shown that the immunogenicity of rodent malaria parasite-derived circumsporozoite protein (CS) can be improved by deleting the glycosyl-phosphatidyl-inositol (GPI) signal sequence. To study whether GPI signal sequence deletion would also improve immunogenicity of CS derived from the major plasmodium species causing mortality in humans (P. falciparum), we tested different variants of the P. falciparum CS protein in the context of a live vector-based vaccine carrier (rAd35). We demonstrate that deletion of the GPI signal sequence from CS did not result in altered expression or secretion. In contrast, cellular localization was clearly altered, which perhaps helps to explain the significant improvement of anti-CS antibody and T-cell responses observed in mice using deletion variants in the context of the rAd35 carrier. Our results show that rational design of antigens is warranted for further development of malaria vaccines.