mRNA vaccine has become a promising technology platform for rabies prevention. This study explores the roles of different structural domains of rabies virus glycoprotein (RABV-G) and heterologous prime-boost strategies for enhanced immune responses and protection. The results suggested that mRNA vaccines encoding full-length RABV-G (RABV-Full) and RABV-R333Q induced strong immune responses and provided full protection against rabies, while mRNA vaccines encoding ectodomain/transmembrane domain (RABV-TE) and ectodomain (RABV-E) were less effective. Heterologous immunization results revealed that mRNA-primed strategies yielded higher long-lasting VNTs, but lower early VNTs than inactivated rabies virus (IRV)-primed strategies. 2×RABV-Full and IRV >
RABV-Full provided 100% protection, while that of RABV-Full>
IRV was 90%. Transcriptome analysis showed that rabies mRNA vaccine induced both MHCI and MHCII antigen presentation, as well as B/T cell activation. In conclusion, full-length RABV-G mRNA vaccines, particularly with an 'IRV prime and RABV-Full boost' strategy, hold great potential for rabies prevention.