The high-rate incidence of hepatic tissue pathologies and lack of enough donors necessitate the development of de novo liver therapeutic protocols and tissue engineering modalities. Due to unique chemical structure, and biological properties, the application of pectin-based hydrogels has been extended in different pathological conditions. Using ozonation [O3 gas (25 mg/h) a flow rate of 1 l/min] for 20, 40, 60, and 80 min, the hepatogenic properties of green synthesized hydrogel containing 6 % oxidized pectin (OP) 1 % collagen (Col) were studied in vitro and acute liver injury mice model. Data confirmed suitable gel content, swelling rate, porosity, Young's modulus, and self-healing capacity of OP-Col hydrogel. Ozonation time can influence the viability and function of human HepG2 cells via the generation-free aldehyde groups in which 40 min-ozonation yielded better biological outcomes (migration↑, and albumin production↑) after neutralization with glycine. The transplantation of glycine-treated liver cells-laden OP-Col hydrogel with 40 min-ozonation protocol reduced the progression of fibrotic changes, and unwanted in situ immune system response in acute liver injury mice model after 14 days. Besides, the systemic levels of albumin and urea were increased coinciding with the reduction of liver cell damage enzymes. OP-based hydrogels can appropriately mimic suitable microenvironments for the regulation of hepatocyte dynamic growth and function in in vitro conditions and following acute injuries. The synthesis of OP-based hydrogel with a specific synthesis protocol promotes the biological activity of transplanted liver cells at the site of injury.