Aim: To study the mechanism of transfection mediated by imidazole-grafted chitosan (CHimi) nanoparticles, to propose new strategies to control and improve the expression of a delivered gene in the context of regenerative medicine. Methods: Biochemical and microscopy methods were used to establish transfection efficiency and nanoparticle intracellular trafficking. The role of CHimi and degree of N-acetylation (DA) on transfection was explored. Results: CHimi was found to promote the expression of a delivered gene during a minimum 7-day period. Additionally, the production of a protein of interest could be upheld by consecutive transfections, without compromising cell viability. Transfection was found to be a time-dependent process, requiring CHimi–DNA complex disassembling. The DA was found to have an impact on transfection kinetics in line with the observation that the rate of lysozyme-mediated nanoparticle degradation increases with the polymer DA. Conclusion: The adjustment of the CH degradation rate can be used as a tool for tuning the expression of a gene delivered by CH-based nanoparticle systems.