Gut injury is a severe and unpredictable illness related to the increased cell death of intestinal epithelial cells (IECs). Excessive IEC apoptotic cell death during the pathophysiological state entails chronic inflammatory diseases. This investigation was undertaken to assess the cytoprotective action and underlying mechanisms of polysaccharides from Tunisian red alga, Gelidium spinosum (PSGS), on H2O2-induced toxicity in IEC-6 cells. The cell viability test was initially carried out to screen out convenient concentrations of H2O2 and PSGS. Subsequently, cells were exposed to 40 µM H2O2 over 4 h in the presence or absence of PSGS. Findings revealed that H2O2 caused oxidative stress manifested by over 70% cell mortality, disturbed the antioxidant defense, and increased the apoptotic rate in IEC-6 cells (32% than normal cells). Pretreatment of PSGS restored cell viability, especially when used at 150 µg/mL and normal cell morphology in H2O2-callenged cells. PSGS also equally sustained superoxide dismutase and catalase activities and hindered the apoptosis induced by H2O2. This protection mechanism of PSGS may be associated with its structural composition. The ultraviolet visible spectrum, Fourier-transformed infrared (FT-IR), X-ray diffraction (XRD), and high-performance liquid chromatography (HPLC) demonstrated that PSGS is mainly sulfated polysaccharides. Eventually, this research work provides a deeper insight into the protective functions and enhances the investment of natural resources in handling intestinal diseases.