This study explored the development of a novel colon-specific drug delivery system for 5-fluorouracil (5-FU) using cross-linked mastic gum (MG) nanoparticles (NPs). The primary goal is to enhance the treatment efficacy of colon cancer while minimizing systemic side effects. We employed Fourier Transform Infrared Radiation (FTIR) and Scanning Electron Microscopy (SEM) for the detailed characterization of the samples. FTIR analysis confirmed the successful cross-linking of MG, whereas SEM images revealed the spherical and uniform morphology of the NPs. Additionally, analysis of drug encapsulation efficiency (83.53%), particle size (240 nm), and drug release kinetics (zero-order), and the drug release percentage (95.20% ) were analyzed. The results demonstrated that MG NPs effectively encapsulated and controlled the release of 5-FU in a colon-targeted manner. This study recommends the proposed drug delivery system because of its potential to improve the outcomes of colon cancer treatment.