Ulcerative colitis (UC), a chronic inflammatory colon disease, often requires long-term care. Pterostilbene (PTB), a naturally occurring substance with strong anti-inflammatory and antioxidant properties, has shown therapeutic potential; however, its poor aqueous solubility and instability at stomach pH limit its clinical application. A colon-targeted drug delivery system loaded with PTB and coated with Eudragit S100 (EU) was developed using mesoporous silica nanoparticles (MSN). The MSN were surface-aminated using 3-aminopropyl triethoxysilane (APTES) to obtain aminated mesoporous silica nanoparticles (AMSN), which provided pH-responsive release and enhanced drug binding. FT-IR spectroscopy confirmed the encapsulation of the drug and amine functionalization through characteristic spectral shifts. X-ray diffraction (XRD) showed decreased crystallinity of PTB, suggesting effective molecular dispersion within the MSN matrix. Differential scanning calorimetry (DSC) was used to confirm the amorphous form of the encapsulated medication. Morphological study using SEM and TEM revealed consistently spherical, porous nanoparticles. Thermogravimetric analysis (TGA) demonstrated the formulation's thermal stability, and Brunauer-Emmett-Teller (BET) analysis showed high surface area and pore volume, both of which are advantageous for efficient drug loading. In vitro release investigations confirmed a pH-dependent, extended release of the drug under intestinal conditions. Additionally, pro-inflammatory cytokines (TNF-α and IL-6) were significantly reduced in the ELISA assays of LPS-stimulated HT-29 cells, demonstrating the formulation's anti-inflammatory efficacy. The development of colon-specific delivery of pterostilbene, the Eudragit-coated PTB-loaded AMSN (Eu-PTB-AMSN), effectively addresses the primary shortcomings of conventional PTB formulations, including low solubility, stomach instability, and systemic adverse effects, while offering a practical therapeutic approach for the treatment of UC.