Polymer-bioceramic composites incorporate the desirable properties of each material while mitigating the limiting characteristics of each component. 1,6-Hexanediol l-phenylalanine-based poly(ester urea) (PEU) blended with hydroxyapatite (HA) nanocrystals were three-dimensional (3D) printed into porous scaffolds (75% porosity) via fused deposition modeling and seeded with MC3T3-E1 preosteoblast cells in vitro to examine their bioactivity. The resulting 3D printed scaffolds exhibited a compressive modulus of ∼50 MPa after a 1-week incubation in PBS at 37 °C, cell viability >95%, and a composition-dependent enhancement of radio-contrast. The influence of HA on MC3T3-E1 proliferation and differentiation was measured using quantitative real-time polymerase chain reaction, immunohistochemistry and biochemical assays. After 4 weeks, alkaline phosphatase activity increased significantly for the 30% HA composite with values reaching 2.5-fold greater than the control. Bone sialoprotein showed approximately 880-fold higher expression and 15-fold higher expression of osteocalcin on the 30% HA composite compared to those of the control. Calcium quantification results demonstrated a 185-fold increase of calcium concentration in mineralized extracellular matrix deposition after 4 weeks of cell culture in samples with higher HA content. 3D printed HA-containing PEU composites promote bone regeneration and have the potential to be used in orthopedic applications.