Dendritic cells (DCs) are potent antigen-presenting cells involved in the initiation and modulation of immune responses after immunization via their ability to process and present antigen to naive T cells. We wanted to examine the role of DCs in the development of protective immunity against nontypeable Haemophilus influenzae (NTHI)-induced experimental otitis media (OM) after intranasal immunization of chinchillas with an NTHI P5-derived synthetic peptide immunogen called LB1. As chinchilla DCs have not been described, we adapted well-established protocols to induce the differentiation of chinchilla bone marrow precursor cells into DCs, which resulted in cells that were morphologically and phenotypically similar to DCs of other species. In vitro, chinchilla DCs readily internalized LB1, upregulated expression of the maturation markers CD80 and major histocompatibility complex class II, and presented processed LB1 to primed CD3+ T cells, which resulted in antigen-specific T-cell proliferation. In vivo, LB1-activated DCs trafficked from the chinchilla nasal cavity primarily to the nasal-associated lymphoid tissues and were detected in close proximity to CD3+ T cells within this lymphoid aggregate. These data are the first to characterize chinchilla DCs and their functional properties. Furthermore, they suggest an important role for chinchilla DCs in the development of protective immunity against experimental NTHI-induced OM after intranasal immunization.