The aim of the present study was to investigate the effects of cobalt nanoparticles (CoNPs) on the proliferation and differentiation of human osteoblasts in vitro, and to investigate the molecular mechanisms via which CoNPs affect proliferation and differentiation of osteoblasts. The MG‑63 human osteoblast cell line was treated with different concentrations of CoNPs for 12 to 48 h in vitro. At each time point, cell morphology was observed and an MTT assay was performed to assess cell viability. Alkaline phosphatase (ALP), osteocalcin (BGLAP), collagen I (COL I), osteoprotegerin (OPG) and receptor activator of nuclear factor κ‑B ligand mRNA expression levels, and ALP, BGLAP and COL protein expression levels, were assessed by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. The viability of MG‑63 cells decreased significantly after treatment with CoNPs. As CoNP concentration increased, a higher growth inhibition and cell death was observed. Compared with CoNPs, treatment with the same concentration of Co2+ may have a greater inhibitory effect on the growth of MG‑63 cells. CoNPs affected the mRNAs expression levels of ALP, BGLAP, COL I and OPG in MG‑63 cells, and reduced the protein expression levels of ALP, BGLAP and COL I. In conclusion, the present study demonstrated that CoNPs induce cytotoxic effects on MG‑63 cells by markedly reducing cell viability and inducing cell death at high concentrations. In addition, CoNPs may inhibit the function and differentiation of osteoblasts by affecting the mRNA and protein expression levels of associated genes. The results of the present study indicate that CoNPs may serve an important role in the aseptic loosening mechanism following total joint replacement surgery, particularly in situations where metal on metal prostheses are used. Further study into inhibiting this effect is required.