Smoking is a risk factor associated with bone and oral diseases, particularly periodontitis. Nicotine, the major toxic component of tobacco, is able to affect the quality and quantity of bone. Osteoblasts serve an important role in bone formation. Thus far, the effects of nicotine on metabolism‑associated gene and protein expression in osteoblasts have been controversial and the mechanisms remain unclear. The present study assessed alterations in osteogenic activity by performing a Cell Counting kit‑8 assay to investigate proliferation, Annexin V‑fluorescein isothiocyanate/propidium iodide staining to investigate apoptosis, alizarin red staining to investigate the formation of mineralized nodules, reverse transcription‑quantitative polymerase chain reaction and western blotting to investigate the mRNA and protein levels of collagen I, alkaline phosphatase, bone osteocalcin, bone sialoprotein and osteopontin; and mRNA microarray expression analysis, Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analysis to investigate the whole genome expression profile of Sprague‑Dawley (SD) rat primary osteoblasts following treatment with different concentrations of nicotine. The results demonstrated that nicotine inhibited proliferation, promoted early apoptosis and inhibited mineralized nodule formation in a dose‑dependent manner by regulating alkaline phosphatase activity and the expression of osteoblast metabolism‑associated genes and proteins. According to microarray analysis, several genes associated with bone metabolism and genes in the Hedgehog and Notch signaling pathways were downregulated significantly in nicotine‑treated osteoblasts. The results of the present study indicated that nicotine may serve an inhibitory, dose‑dependent role in SD rat primary osteoblasts that may be caused by the perturbation of genes and signaling pathways associated with bone formation. These results may provide a theoretical basis for future research regarding bone metabolism and targeted treatment of oral diseases associated with smoking.