Proton pump inhibitors (PPIs) are a class of drugs that irreversibly inhibit the H+/K+-ATPase in gastric parietal cells. Since an association between PPI use and increased fracture risk has been found, the aim of this study was to detect potential adverse effects of pantoprazole, a representative of the PPIs, on primary human osteoblasts in vitro. The isolated cells were stimulated with pantoprazole concentrations ranging from 0μg/ml to 10μg/ml. Changes in proliferation, total cell number, viability, cytotoxicity, alkaline phosphatase activity, total protein synthesis and gene expression on mRNA level were determined over a period of 7 days. Pantoprazole stimulation resulted in increased viability and decreased cytotoxicity in the osteoblasts. The proliferation rate was stable and so was the relative cell number. Only at the highest pantoprazole concentration on day 7, a slight decrease of the cell number was detected. Alkaline phosphatase activity increased over the tested period under exposure to pantoprazole (p<0.05 at 3μg/ml and 10μg/ml pantoprazole). Osteoblast-specific gene expression was increased through pantoprazole stimulation compared to the control on day 3. Towards day 7, gene expression returned to baseline levels or decreased slightly compared to unexposed cells. Interestingly, this in vitro experiment detected no evidence of adverse effects of PPIs on primary human osteoblasts. Osteoblasts were rather more viable with increased mitochondrial activity, gene expression and protein synthesis under pantoprazole stimulation. Therefore, these in vitro results do not suggest that impaired osteoblast function is the cause of an increased fracture risk in patients under PPI therapy.