Purpose <p>The study aimed to create functionalized modified 5-fluorouracil hyaluronic acid microspheres (PAR-HA Mps/5-Fu) for localized drug delivery to colorectal cancer by targeting the CD44 receptor, with the ability to control release rate by different PH release environments.</p> Methods <p>PAR-HA Mps/5-Fu were prepared by emulsification cross-linking method. The optimal oral formulation was identified through a combination of single-factor examination and orthogonal experimental design. In vitro release experiments were performed in different media and drug release models were fitted. The in vivo pharmacokinetic profile, in vivo safety, and in vivo biodistribution of PAR-HA Mps/5-Fu were investigated in in vivo examinations.</p> Results <p>In this study, PAR-HA Mps/5-Fu was successfully prepared. While PAR-HA Mps/5-Fu was difficult to dissolve (16.17%) in artificial gastric fluid, it was constantly released (98.45%) in artificial colonic fluid, and its release pattern fit the Weibull equation. The results of the in vivo experiments showed that PAR-HA Mps/5-Fu had good drug localization in the pathological environment, the pharmacokinetic parameters of the formulation showed significant improvement compared to the 5-FU solution. Meanwhile, oral administration of PAR-HA Mps/5-Fu did not affect the organism, tissues, or blood system.</p> Conclusion <p>The study demonstrated that PAR-HA Mps/5-FU can facilitate the local delivery of 5-FU to the colon, thereby addressing the clinical limitations of 5-FU, including its short plasma half-life, low bioavailability, and high toxicity.</p> Graphical Abstract <p></p>