Ulcerative colitis (UC) is a chronic inflammatory disorder with a complex etiology, characterized by intestinal inflammation and barrier dysfunction. Platycodon grandiflorus polysaccharides (PGP), the primary component of Platycodon grandiflorus, and hesperidin (Hesp), a prominent active component in Citrus aurantium L. (CAL), have both demonstrated anti-inflammatory properties. This study aims to elucidate the underlying mechanism of the synergistic effect of PGP combined with Hesp on UC, focusing on the coordinated interaction between the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathways. A mouse model of UC induced by dextran sulfate sodium (DSS) and a cell model using lipopolysaccharide (LPS)-induced RAW264.7/IEC6 cells were employed to investigate the in vitro and in vivo anti-inflammatory effects of PGP combined with Hesp on UC and its potential mechanism of action. The results indicated that compared to the effects of either drug alone, the combination of PGP and Hesp significantly modulated inflammatory factor levels, inhibited oxidative stress, regulated colonic mucosal immunity, suppressed apoptosis, and restored intestinal barrier function in vitro and in vivo. Further in vitro studies revealed that PGP significantly inhibited the PI3K/AKT signaling pathway, while Hesp significantly inhibited the JAK2/STAT3 signaling pathway. The use of inhibitors and activators targeting both pathways validated the synergistic effects of PGP combined with Hesp on the PI3K/AKT and JAK2/STAT3 signaling pathways. These findings suggest that PGP combined with Hesp exhibits a synergistic effect on DSS-induced colitis, potentially mediated through the phosphatase and tensin homolog (PTEN)/PI3K/AKT and interleukin-6 (IL-6)/JAK2/STAT3 signaling pathways.