Self-assembling peptide nanofibers have been shown to have self-adjuvanting properties—stimulating targeted immune responses in vivo and raising long lasting antibody responses without an accompanying inflammatory response. Despite these novel properties, the mechanisms behind nanofiber induced immune responses are still not known. To investigate these mechanisms, the self assembling Q11 peptide sequence was used. Dendritic cell (DC) activation was measured in vitro using the DC2.4 dendritic cell line, which immunohistochemistry identified nanofiber localization within the lymph node in a murine model of immunization. DC activation in vitro was shown to be dependent on the presence and concentration of the Pan-DR-epitope (PADRE) within the nanofiber. Measurements made by flow cytometry showed a dose dependent upregulation of CD83 and MHCII when PADRE containing nanofibers were added to DC cell culture media. Additionally, immunohistochemical analysis of lymph nodes containing nanofibers conjugated with the fluorescent molecule Tetramethyl-6-Carboxyrhodamine (TAMRA) showed a colocalization of injected nanofibers with both macrophages and DCs in vivo. These results indicate that nanofiber induced immune responses function in both canonical pathways, involving DCs, and non-standard pathways, involving macrophages. This provides a framework to begin to understand the complex immune responses generated by self-assembled peptide nanofiber vaccines.