Mucosal vaccines are powerful tools for combatting emerging infectious diseases, particularly mucosal-associated pathogens. However, one of the main bottlenecks in developing mucosal vaccines is the lack of accurate animal models. In this study, a vesicular stomatitis virus (VSV)-vectored Middle East respiratory syndrome coronavirus (MERS-CoV) mucosal vaccine was designed for investigations. Compared with the VSV backbone, rVSVΔG-MERS-S exhibited altered cellular tropism, as determined by MERS-S. In wild-type (WT) C57BL-6J mice and hamsters, the nasal spray of rVSVΔG-MERS-S was poorly immunogenic. In contrast, rVSVΔG-MERS-S was highly immunogenic in transgenic mice (hDPP4 mice) and hDPP4-transduced hamsters harboring the functional MERS-CoV receptor. Compared with those of WT C57BL-6J mice, the nasal spray of rVSVΔG-MERS-S resulted in effective antigen-presenting cell (APC) priming, Tfh-GcB-plasma cell (pC) proliferation, and robust humoral and cellular responses, together with the activation of antiviral signaling pathways in hDPP4 mice. Similarly, rVSVΔG-MERS-S was highly immunogenic in alpacas and rhesus monkeys, which are naturally susceptible to MERS-CoV and harbor the effective DPP4 receptor. The alignment of hDPP4 receptors in these animals revealed that L294, I295, and R336 in DPP4 are key residues contributing to differences in sensitivity across species. Consistently, a high binding affinity was observed between human, alpacas, and rhesus monkey DPP4 receptors and MERS-CoV receptor binding domains (RBDs) compared with that of mice and hamster. Overall, this proof-of-concept study not only guides the selection of appropriate animal models for the evaluation of mucosal vaccines of MERS but also provides evidence that functional receptors DPP4 in animal models are prerequisites for the immune performance of the MERS mucosal vaccine.