The objective of this study was to investigate the in vitro and in vivo effects of blank chitosan nanoparticles on various molecular markers such as nitric oxide (NO) production, IL-6 gene expression, and lymphocyte proliferation involved in the wound healing process. In addition, the membrane effects of chitosan nanoparticles were evaluated using phospholipid vesicles as a model membrane. Peripheral blood mononuclear cells (PBMC) were treated with blank chitosan nanoparticles, and the effect on NO production, IL-6 gene expression, and lymphocyte proliferation was evaluated. It was observed that IL-6 gene expression was not induced at any of the doses used; however, a statistically significant dose-dependent increase in NO production was observed at doses above 68.18 μg/mL equivalent to chitosan. Furthermore, chitosan nanoparticles showed a statistically significant and dose-dependent lymphocyte proliferation as compared to the control (P < 0.05). It was observed that blank chitosan nanoparticles resulted in strong membrane perturbation when evaluated by differential scanning calorimetry studies. The in vivo effects of the blank chitosan nanoparticles were evaluated using a wound healing model. Blank chitosan nanoparticles showed significantly higher NO production in vivo as compared to the control. Overall, the study clearly indicates the immunoactivating nature of chitosan nanoparticles and their strong membrane interactive potential.