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Emerging evidence indicates that microRNAs (miRNAs) have important roles in regulating osteogenic differentiation and bone formation. Thus far, no study has established the pathophysiological role for miRNAs identified in human osteoporotic bone specimens. Here we found that elevated miR-214 levels correlated with a lower degree of bone formation in bone specimens from aged patients with fractures. We also found that osteoblast-specific manipulation of miR-214 levels by miR-214 antagomir treatment in miR-214 transgenic, ovariectomized, or hindlimb-unloaded mice revealed an inhibitory role of miR-214 in regulating bone formation. Further, in vitro osteoblast activity and matrix mineralization were promoted by antagomir-214 and decreased by agomir-214, and miR-214 directly targeted ATF4 to inhibit osteoblast activity. These data suggest that miR-214 has a crucial role in suppressing bone formation and that miR-214 inhibition in osteoblasts may be a potential anabolic strategy for ameliorating osteoporosis.