Silver (Ag) possesses potent antimicrobial properties and is used as a coating for medical devices. The impact of silver ions released from orthopedic implants on the differentiation and osteoid formation of different osteogenic cells has yet to be systematically studied. In the present study, human mesenchymal stem cells (hMSCs) and primary human osteoblasts (hOBs) were exposed to different static Ag+ concentrations (0, 0.5, 1.0 or 1.5 ppm) or dynamic Ag+ concentrations (range 0 to 0.7 ppm) that simulated the temporal release pattern from a Ag-nitrate coating of trabecular titanium (TLSN). Cell morphology was investigated by phase contrast and fluorescence microscopy. The activities of alkaline phosphatase (ALP) and lactate dehydrogenase, osteogenic gene expression (COL1A1, COL1A2 and ALPL), and osteoid deposition were examined for up to 4 weeks. DAPI and carboxyfluorescein diacetate staining revealed changes in the morphology of hOBs treated with ≥0.5 ppm Ag+, while osteocalcin-positive cells were observed primarily in the untreated group. Elevated Ag+ concentrations did not impact the production of ALP by either hMSCs or hOBs. Treatment with 1.5 ppm Ag+ or TLSN Ag+ led to a modest reduction in COL1A2 and ALPL levels in hMSCs at 2 weeks but not at 4 weeks nor in hOBs. In hMSC cultures, mineralization decreased at ≥1 ppm Ag+, whereas the same concentration range significantly reduced mineralization in hOB cultures. In conclusion, Ag+ concentrations ranging from 1.0 to 1.5 ppm may interfere with osteogenic differentiation, possibly by altering gene expression, thereby affecting mineralization. Only Ag+ concentrations up to 0.5 ppm allowed undisturbed osteogenic differentiation and mineralization. These findings pertain to creating Ag coatings of titanium intended for cementless fixation into host bone.