Fracture healing is a complex process in which the periosteum and endosteum become the main sources of osteoblast progenitor cells. However, cellular mechanisms and signaling cascades underlying the early stages of osteoblast progenitors differentiation in adult bone are still not well understood. Therefore, we performed shotgun proteomics analysis of primary culture of isolated human osteoblasts from femur of adult donors in undifferentiated conditions and on the fifth day of osteogenic differentiation in vitro. This is an early timepoint in which we have observed no extracellular matrix mineralization yet. 1612 proteins identified with at least two unique peptides were included in proteomics analysis. Data are available via ProteomeXchange with identifier PXD033697. Despite the fact, that matrix mineralization starts only after induction of osteogenic differentiation, we revealed unexpectedly weak physiological shift associated with a decrease of cells proliferative activity and changes in proteins involved in extracellular matrix secretion and organization. We demonstrated that osteoblasts were positive for markers of later osteogenic differentiation stages during standard cultivation: osteopontin, osteocalcin, BMP-2/4 and RUNX2. Therefore, further differentiation required for matrix mineralization needs minimal physiological changes.