In this paper we investigated a novel and non-invasive approach for an endogenous osteoblast stimulation mediated by boron nitride nanotubes (BNNTs). Specifically, following the cellular uptake of the piezoelectric nanotubes, cultures of primary human osteoblasts (hOBs) were irradiated with low frequency ultrasound (US), as a simple method to apply a mechanical input to the cells loaded with BNNTs. This in vitro study was aimed at investigating the main interactions between hOBs and BNNTs and to study the effects of the 'BNNTs + US' stimulatory method on the osteoblastic function and maturation.A non-cytotoxic BNNT concentration to be used in vitro with hOB cultures was established. Moreover, investigation with transmission electron microscopy/electron energy loss spectroscopy (TEM/EELS) confirmed that BNNTs were internalized in membranal vesicles. The panel of investigated osteoblastic markers disclosed that BNNTs were capable of fostering the expression of late-stage bone proteins in vitro, without using any mineralizing culture supplements. In our samples, the maximal osteopontin expression, with the highest osteocalcin and Ca(2+) production, in the presence of mineral matrix with nodular morphology, was observed in the samples treated with BNNTs + US. In this group was also shown a significantly enhanced synthesis of TGF-β1, a molecule sensitive to electric stimulation in bone. Finally, gene deregulations of the analyzed osteoblastic genes leading to depletive cellular effects were not detected. Due to their piezoelectricity, BNNT-based therapies might disclose advancements in the treatment of bone diseases.