Several approaches to combine bone substitutes with biomolecules, cells or mechanical loading have been explored as an alternative to the limitation and risk-related bone auto- and allo-grafts. In particular, human bone progenitor cells seeded in porous poly(L-lactic acid)/tricalcium phosphate scaffolds have shown promising results. Furthermore, the application of mechanical loading has long been known to be a key player in the regulation of bone architecture and mechanical properties. Several in vivo studies have pointed out the importance of its temporal offset. When an early mechanical loading was applied a few days after scaffold implantation, it was ineffective on bone formation, whereas a delayed mechanical loading of several weeks was beneficial for bone tissue regeneration. No information is reported to date on the effectiveness of applying a mechanical loading in vivo on cell-seeded scaffold with respect to bone formation in a bone site. In our study, we were interested in human bone progenitor cells due to their low immunogenicity, sensitivity to mechanical loading and capacity to differentiate into osteogenic human bone progenitor cells. The latest capacity allowed us to test two different bone cell fates originating from the same cell type. Therefore, the general aim of this study was to assess the outcome on bone formation when human bone progenitor cells or pre-differentiated osteogenic human bone progenitor cells are combined with early and delayed mechanical loading inside bone tissue engineering scaffolds. Scaffolds without cells, named cell-free scaffold, were used as control. Surprisingly, we found that (1) the optimal solution for bone formation is the combination of cell-free scaffolds and delayed mechanical loading and that (2) the timing of the mechanical application is crucial and dependent on the cell type inside the implanted scaffolds.