Hypoxia has been implicated in the pathogenesis of many bone-related diseases but its effects on osteoblasts are not fully understood. This study aimed to investigate the effect of short-term hypoxia on cell viability and collagen secretion in human osteoblast-like SaOs-2 cells in vitro. Hypoxia was induced by placing the cells in a multigas chamber containing 92%N2, 5%CO2 and 3%O2 at 37°C for four hours. The control cultures were incubated at 95% O2 and 5% CO2 at 37°C. Following hypoxic exposure, there was an enhanced immunoexpression of hypoxia-inducible factor-1α (HIF-1α) in SaOs-2 cells as compared to normoxic control cells, suggesting the presence of hypoxia. Along with that, the release of total reactive oxygen species/reactive nitrogen species (ROS/RNS) was significantly increased by hypoxic SaOs-2 cells. Western blotting showed significant upregulation of the redox-sensitive proteins endoplasmic oxidoreductin-1 (Ero1-Lα) and protein disulfide-isomerase (PDI), which are implicated in oxidative protein folding. The level of hydroxyproline, as measured by colorimetric assay, was significantly reduced in culture medium of hypoxic SaOs-2 cells, which suggests that hypoxia may alter collagen secretion. In addition to that, the viability of SaOs-2 cells was affected by hypoxia as shown by trypan-blue exclusion text, viability assay and a significant increase in TUNEL-positive cells. In conclusion, our results suggest that hypoxia affects the viability as well as the function of osteoblast-like SaOs-2 cells in vitro as shown by the reduction in hydroxyproline content, the presence of oxidative stress, aberrant protein folding and cell death.