In our previous studies, randomly 50% N-acetylated low molecular weight chitosan (LMWC) has been confirmed as a potential carrier for the site-specific delivery of prednisolone to kidney, suggesting specific uptake of LMWC in kidney. Interestingly, aminoglycoside, a well-known ligand of megalin receptor, shares a similar glucosamine unit level with LMWC. Based on these, we proposed that the specific renal uptake of LMWC might also be mediated by the megalin receptor. To test this hypothesis, we performed the present study to further investigate the renal uptake process of LMWC and its possible mechanism as well. First, LMWC was found by fluorescent microscopy to selectively accumulate in the kidneys, especially in the renal proximal tubules after iv injection in mice. Then, our research also revealed that LMWC was internalized into renal tubular cells (RTCs) through endocytic pathway, with a concentration-dependent and saturable pattern. The uptake of LMWC could be competitively inhibited in the presence of gentamycin, a kind of aminoglycosides. In addition, cytotoxicity assay showed that there were no obvious effects of LMWC on the viability of L929 and RTCs lines. Finally, megalin-shedding mouse models were established and the distribution of LMWC in tissues of normal and megalin-shedding mice was evaluated. Consistent with gentamycin inhibition assay, in vivo results also suggested the role of megalin in the uptake of LMWC in kidney. In conclusion, LMWC could be specifically taken up by RTCs, where the megalin receptor would likely mediate its binding and uptake.