Abstract In the event of excessive damage to bone tissue, the self-healing process alone is not sufficient to restore bone integrity. Three-dimensional (3D) printing, as an advanced additive manufacturing technology, can create implantable bone scaffolds with accurate geometry and internal architecture, facilitating bone regeneration. This study aims to develop and optimise hydroxyapatite-polyethylene glycol diacrylate (HA-PEGDA) hydrogel inks for extrusion 3D printing of bone tissue scaffolds. Different concentrations of HA were mixed with PEGDA, and further incorporated with pluronic F127 (PF127) as a sacrificial carrier. PF127 provided good distribution of HA nanoparticle within the scaffolds and improved the rheological requirements of HA-PEGDA inks for extrusion 3D printing without significant reduction in the HA content after its removal. Higher printing pressures and printing rates were needed to generate the same strand diameter when using a higher HA content compared to a lower HA content. Scaffolds with excellent shape fidelity up to 75-layers and high resolution (∼200 µm) with uniform strands were fabricated. Increasing the HA content enhanced the compression strength and decreased the swelling degree and degradation rate of 3D printed HA-PEGDA scaffolds. In addition, the incorporation of HA improved the adhesion and proliferation of human bone mesenchymal stem cells (hBMSCs) onto the scaffolds. 3D printed scaffolds with 2 wt% HA promoted osteogenic differentiation of hBMSCs as confirmed by the expression of alkaline phosphatase activity and calcium deposition. Altogether, the developed HA-PEGDA hydrogel ink has promising potential as a scaffold material for bone tissue regeneration, with excellent shape fidelity and the ability to promote osteogenic differentiation of hBMSCs.