The immunogenicity of current human immunodeficiency virus-1 (HIV-1) canarypox vaccines is weak and needs to be improved. Ligation of OX40 (CD134), a member of tumor necrosis factor receptor superfamily (TNFRSF), by its ligand OX40L (CD252), a tumor necrosis factor superfamily (TNFSF) molecule, has been demonstrated to provide a pivotal costimulatory signal to enhance CD4(+) T cell help of humoral and cytotoxic T cell immune responses. The present study examined whether an OX40L-expressing vector could boost the immunogenicity of the HIV-1 canarypox vaccine, vCP1452, in mice. Co-immunization of mice with OX40L-expressing canarypox and vCP1452 augmented HIV-1 specific CD8(+) T cell responses in terms of frequency and cytokine expression. OX40L-expressing canarypox enhanced the frequency of antigen specific CD8(+) T cells with an effector (CD127(-)CD62L(-)) phenotype, which was associated with an ex vivo expansion of HIV-1 specific CD4(+) T cells. This was in contrast to our previous work in which a CD40L-expressing construct preferentially enhanced antigen specific memory responses [Liu J, Yu Q, Stone GW, Yue FY, Ngai N, Jones RB, et al. CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals. Vaccine 2008;26(32):4062-72]. Surprisingly, OX40L did not enhance antibody responses elicited by the HIV-1 canarypox vaccine. We saw no added benefit by combining OX40L and CD40L vectors as an adjuvant strategy for vCP1452. Our results indicate that, similar to CD40L, canarypox vectors expressing OX40L can enhance the cellular but not humoral immunogenicity of HIV-1 canarypox vaccines. In summary, our findings show that OX40L can be used as a molecular adjuvant to enhance T cell immune responses.