Background: Plecanatide, which is used to treat irritable bowel syndrome constipation (IBS-C), is highly susceptible to gastric enzymatic degradation, impairing critical therapeutic efficacy and bioavailability. This work designed an improved site-directing, colon-targeted mucoadhesive formulation to guarantee site-specific delivery and efficacy. Methods: Design-Expert software 13 was used in a systematic optimization system with different concentrations of Na-alginate, Carbopol 934, and Methocel K15M. Flowability and compressibility were evaluated by pre-compression analysis. To specifically target the distal colon, the in vitro dissolution kinetics and cellular activity against the HCT116 colorectal carcinoma cells were assessed. They were in vivo studies that measured mucosal recovery, submucosal pathology, and systemic oxidative stress biomarkers (MDA and SOD). In the accelerated stability testing, the standardized periods were tested under controlled laboratory conditions. Results: Methocel K15M proved to be the best polymer. The formulation exhibited 24 h sustained release, which fit best in the Hixson–Crowell (r = 0.980) and Higuchi (r = 0.956) models. Cellular activity against the HCT116 cells was high (IC50 = 79.31 ug/mL), being ten-fold better than the raw API. In vivo, the mucoadhesive system demonstrated almost total inflammatory resolution of all the layers of the colon. As opposed to regular tablets, it had a tremendous impact in reducing MDA (p = 0.014) and raising the activity of SOD (p = 0.002). Accelerated stability testing (40 °C/75% RH for 6 months) showed a slight decrease in drug content from 100.85% to 96.78%, which remained within the acceptable pharmacopeial limits (90–110%), confirming formulation stability. Conclusions: The mucoadhesive Plecanatide formulation is a “site-directing colon-targeted mucoadhesive formulation” that can effectively overcome the shortcomings of traditional therapy; i.e., it exhibits the potential for better compliance. This formulation mitigates inflammatory and oxidative stress, offering a stable, promising alternative for managing chronic IBS-C symptoms and enhancing patient health outcomes in contemporary clinical practice and pharmaceutical standards.