N16, a nacreous protein isolated from Pinctada martensii, is related to nacreous layer formation. Our previous study indicated that N16 showed dual regulatory effects by inducing osteoblast biomineralization as well as inhibiting osteoclast formation. In order to obtain large quantity of N16 for animal experiment and clinical trial, a fermentation and preparative purification method was established. The N16 cDNA was cloned to a BL21(DE3)plysE-pET32a vector and grown in a 20 L fermenter. The medium, temperature, pH and dissolved oxygen (DO) were optimized. N16 was expressed in inclusion bodies. It was denatured and refolded in 8 M urea buffer and purified to 97% purity by passing through a gel filtration column. The glucocorticoid induced osteoporosis (GIO) rat model was used to investigate the anti-osteoporosis activity of N16 in vivo. Results showed that the decrease of the bone mineral density (BMD) and the ultimate load was significantly relieved after N16 treatment. N16 displayed dual regulatory effects by promoting osteogenesis as well as inhibiting bone resorption in vivo. Our work will contribute to further clinical studies on N16 for osteoporosis treatment.