Overall, the reduced or increased sensitivity to Ca2+ of four out of five OI-associated PLS3 mutations characterized in the present study as well as their respectively perturbed localization in osteoblast, osteocyte, and fibroblast cells indicate that the delicately controlled Ca2+-dependent regulation of actin bundling by PLS3 is essential for normal bone formation. Here, PLS3 is linked to osteogenesis imperfecta.