A comparative study was performed to investigate the ultrastructural and biomolecular properties of osteoblasts induced by three types of single-walled carbon nanotubes (SWNTs). The results on cellular uptake and ultrastructural alteration indicate that SWNTs enter osteoblasts by endocytosis. SWNTs-COOH and SWNTs-OH particles were freely dispersed in the cytoplasm, while pristine SWNTs were localized to the periphery of the cell. Both SWNTs-OH and SWNTs-COOH promoted cell changes in cell activity regarding mRNA expression at doses of 50 and 100 μg/mL in the first 24 h. When treated with 50 μg/mL SWNTs-COOH for 48 h, the expression of type I collagen increased by 6.3-fold (for MG63) or 9.1-fold (for primary osteoblasts) compared with the control group. The present study observed for the first time that SWNTs-COOH initiated the prompt and the maximum upregulation of type I collagen gene expression, and simultaneously induced the expansion of the endoplasmic reticulum for increased protein synthesis, which in turn accelerated the mineralization process. However, impaired cell properties and mitochondrial injury were detected following treatment with SWNTs at 100 μg/mL after 48 h. In conclusion, we believe that SWNTs-COOH is a good candidate for the fabrication of biomedical scaffolds for bone regeneration.