Chitosan and its derivatives are widely used in vaccine adjuvants and delivery systems. Vaccine antigens encapsulated in or conjugated onto N-2-hydroxypropyl trimethyl ammonium chloride chitosan/N,O-carboxymethyl chitosan nanoparticles (N-2-HACC/CMCS NPs) induce strong cellular, humoral, and mucosal immune responses, but the mechanism of action is not fully understood. Therefore, the purpose of this study was to explore the molecular mechanism of composite NPs by upregulating the cGAS-STING signalling pathway to enhance the cellular immune response. We showed that the N-2-HACC/CMCS NPs could be taken up by RAW264.7 cells and produced high levels of IL-6, IL-12p40, and TNF-α. The N-2-HACC/CMCS NPs activated BMDCs, promoted Th1 responses, and enhanced the expression of cGAS, TBK1, IRF3, and STING, as further demonstrated by qRT–PCR and western blotting. Moreover, the NP-induced expression of I-IFNs, IL-1β, IL-6, IL-10 and TNF-α in macrophages was closely related to cGAS-STING. These findings provide a reference for chitosan derivative nanomaterials as vaccine adjuvants and delivery systems and demonstrate that N-2-HACC/CMCS NPs can engage the STING-cGAS pathway to trigger the innate immune response.