It is acknowledged that low molecular weight chitosan (LMWC) is advantageous over high molecular weight chitosan (HMWC) in the biodegradability. In this report, the potential of LMWC in DNA vaccine delivery via mucosa was evaluated. Firstly, the effects of molecular weight of chitosan on the physicochemical properties and in vitro transfection efficiency of chitosan/DNA polyplexes were investigated. Secondly, the capabilities of the polyplexes based on LMWC to elicit serum IgG antibodies and to attenuate the development of atherosclerosis after intranasal vaccination were compared with the polyplexes based on HMWC in the rabbit model. Finally, the intramucosal transport of the double-labeled polyplexes was observed by confocal microscopy. The results indicated that LMWC had lower binding affinity to DNA and mediated higher transfection efficiency. Intranasal vaccination with LMWC/DNA polyplexes could elicit significant systemic immune responses, modulate the plasma lipoprotein profile and attenuate the progression of atherosclerosis. Those aspects were comparable to those obtained by HMWC/DNA polyplexes. As revealed by confocal images, LMWC/DNA polyplexes remained stable during interaction with the nasal mucosa, and were internalized by nasal epithelial cells, which was similar to the case of HMWC/DNA polyplexes. In conclusion, LMWCs have potential applications in DNA vaccine delivery via mucosa.