Chitosan, a natural, biodegradable, and biocompatible polymer derived from chitin, has garnered significant attention as a promising carrier for gene and RNA delivery in therapeutic applications. Its unique properties, including cationic charge, mucoadhesive properties, and biodegradability, make it an ideal candidate for non-viral gene delivery systems. This chapter explores the mechanisms of chitosan-based gene and RNA delivery, highlighting the interactions between chitosan and nucleic acids, as well as the role of chitosan in cellular uptake and intracellular trafficking. Furthermore, we discuss the various strategies employed to enhance the efficiency and specificity of chitosan-based delivery systems, such as chemical modifications, nanoparticle formulation, and combination with other polymers or targeting ligands. The therapeutic potential of chitosan-based gene and RNA delivery is vast, encompassing a wide range of diseases. These include genetic disorders, cancer, infectious diseases, and vaccine development. By leveraging the advantages of chitosan, researchers are developing innovative therapeutic approaches that hold promise for improving patient outcomes.