Objective · To construct a natural polymers-based hydrogels with enhanced mechanical property through double network (DN) strategy under the premise of retaining the good biocompatibility of natural polymers, and then evaluate the beneficial effect of hydrogels on the osteogenic differentiation of rat bone mesenchymal stem cells (rBMSCs). Methods · Methacrylated hyaluronic acid (HAMA) and methacrylated natural gelatin (GelMA) were synthesized through reaction of HA and Gel with methacrylic anhydride (MA), respectively. Thereafter, HA-Gel DN hydrogels were fabricated through two-step photo-crosslinking. The basic physicochemical properties of hydrogels were evaluatedscanning electron microscopy, swelling, compression and degradation analysis. Hydrogels were applied as substrate materials for rBMSCs culture in vitro. Cell viability, attachment, spreading and proliferation were assessedCCK-8 analysis and fluorescent staining analysis. The osteogenic differentiation of rBMSCs was determinedquantitative PCR and Western blotting analysis. Results · In comparison to HA hydrogels and Gel hydrogels, HA-Gel DN hydrogels showed more suiphysicochemical properties, such as more suiwater absorption and water retention [(12.6± 0.7) fold, (10.3± 0.4) fold], stronger mechanical property [(43.7± 5.6) kPa] and slower degradation rate[(82.3±3.9)%for12weeks] for osteogenic differentiation of rBMSCs. Experiment in vitro revealed that HA-Gel DN hydrogels had good biocompatibility, quantitative PCR revealed that it could promote the of osteogenic genes including Runx2, BSP, OPN, OCN, OSX and ALP. Western blotting revealed that the HA-Gel DN hydrogels also increased the levels of osteogenic proteins (OPN, OSX and BSP). Conclusion · HA-Gel DN hydrogels have good biocompatibility and promote the osteogenic differentiation of rBMSCs, which provide a new experimental basis for DN hydrogels becoming the potential material for bone defects repair.