The objective of this study was to investigate whether the immunostimulatory properties of human monocyte-derived dendritic cells (DCs) could be enhanced by triggering OX40/OX40L signaling. Since monocyte-derived DCs possess only low-cell surface levels of OX40L in the absence of CD40 signaling, OX40L was expressed by transfection of DCs with the corresponding mRNA. We show that OX40L mRNA transfection effectively enhanced the immunostimulatory function of DCs at multiple levels: OX40L mRNA transfection augmented allogeneic and HLA class II epitope-specific CD4⁺ T-cell responses, improved the stimulation of antigen-specific cytotoxic T lymphocytes (CTLs) in vitro without interfering with the prostaglandin E₂ (PGE₂)–mediated migratory function of the DCs, and facilitated interleukin 12 p70 (IL-12p70)–independent T helper type 1 (Th1) polarization of naive CD4⁺ T-helper cells. Furthermore, vaccination of tumor-bearing mice using OX40L mRNA–cotransfected DCs resulted in significant enhancement of therapeutic antitumor immunity due to in vivo priming of Th1-type T-cell responses. Our data suggest that transfection of DCs with OX40L mRNA may represent a promising strategy that could be applied in clinical immunotherapy protocols, while circumventing the current unavailability of reagents facilitating OX40 ligation.