Large volume bone defects that do not spontaneously heal despite surgical stabilization ("critical-sized" defects) remain a challenge to treat clinically. Recent research investigating bone regenerative implants made from 3D printed materials have shown promise as a potential alternative to current treatment methods, such as autografting, allografting, and multi-step surgical interventions. Recent work has shown that implanting 3D printed calcium phosphate cement (CPC) scaffolds loaded with bone morphogenetic protein-2 (BMP-2) can provide a one-step surgical intervention that has similar bone healing outcomes to a popular two-step intervention: the Masquelet technique. The aim of this study was to investigate whether a 3D printed CPC scaffold loaded with a lyophilized polyplex gene-delivery formulation could serve as an alternative to loading BMP-2 protein onto such scaffolds. We 3D printed CPC scaffolds, hardened them with multiple methods, and explored the impact of these hardening methods on surface texture, mechanical strength, osteogenic differentiation, and ion flux. We then gene-activated these materials with cationic polyplexes containing plasmid DNA encoding reporter genes to investigate transfection from the gene-activated scaffolds. We found that incubating CPC scaffolds in aqueous solutions after initial hardening in a humid environment could enhance scaffold mechanical strength (compressive strength of 21.28 MPa vs. 6.54 MPa) and osteogenic differentiation. We also found that when we increased the total surface area of the CPC material exposed to polyplex solutions, there was a reduction in transfection via adsorption of polyplexes to the CPC surface. This study shows that 3D printed, gene-activated CPC scaffolds are a promising avenue for future exploration in the field of bone regeneration, though the level of gene expression induced by the scaffolds must be improved.