Cells respond to a wide range of mechanical stimuli such as fluid shear and strain, although the contribution of gravity to cell structure and function is not understood. We hypothesize bone-forming osteoblasts are sensitive to increased mechanical loading by hypergravity. A centrifuge suitable for cell culture was developed and validated, then primary cultures of fetal rat calvarial osteoblasts at various stages of differentiation were mechanically loaded by hypergravity. We measured microtubule network morphology as well as release of the paracrine factor prostaglandin E₂ (PGE₂). In immature osteoblasts, a stimulus of 10 times gravity (10-g) for 3 hr increased PGE₂ (2.5 fold) and decreased microtubule network height (1.12 fold), without affecting cell viability. Hypergravity (3 hr) caused dose-dependent (5-g to 50-g) increases in PGE₂ (5.3-fold at 50-g) and decreases in microtubule network height (1.26-fold at 50-g). PGE₂ release depended on duration but not orientation of the hypergravity load. As osteoblasts differentiated, sensitivity to hypergravity declined. We conclude that primary osteoblasts demonstrate dose- and duration-dependent sensitivity to gravitational loading which appears blunted in mature osteoblasts.