AbstractMouse splenic dendritic cell (DC) subsets possess distinct antigen‐presentation abilities. CD8+ DC are specialized in cross‐presentation of antigens to CD8+ T cells, whereas CD8– DC are more efficient in antigen presentation to CD4+ T cells. In this study, we examined the capacity of CD8+ and CD8– DC subsets to present fungal antigens in MHC class I and II molecules to CD8+ and CD4+ T cells, respectively. We used ovalbumin‐expressing Saccharomyces cerevisiae (yeast‐OVA) as a fungal model system. Both CD8+ and CD8– DC subsets phagocytosed yeast in equal amounts and uptake was mediated via dectin‐1. In addition, both DC subsets induced similar OVA‐specific CD4+ T cell proliferation after incubation with yeast‐OVA. However, the induction of OVA‐specific CD8+ T cell activation was largely restricted to the CD8– DC subset. Furthermore, only CD8– DC produced cytokines such as IL‐10 and TNF‐α and increased IL‐23p19 and IL‐23p40 mRNA levels in response to yeast. Our results strongly suggest that DC subsets have different functions in the elicitation of adaptive immune responses in vivo.