Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a glycosaminoglycan (GAG)- and collagen-binding anchor protein highly expressed in cartilage, basement membranes, and developing bone. We observed that PRELP inhibited in vitro and in vivo mouse osteoclastogenesis through its GAG-binding domain ((hbd)PRELP), involving (a) cell internalization through a chondroitin sulfate- and annexin II-dependent mechanism, (b) nuclear translocation, (c) interaction with p65 nuclear factor kappaB (NF-kappaB) and inhibition of its DNA binding, and (d) impairment of NF-kappaB transcriptional activity and reduction of osteoclast-specific gene expression. (hbd)PRELP does not disrupt the mitogen-activated protein kinase signaling nor does it impair cell survival. (hbd)PRELP activity is cell type specific, given that it is internalized by the RAW264.7 osteoclast-like cell line but fails to affect calvarial osteoblasts, bone marrow macrophages, and epithelial cell lines. In vivo, (hbd)PRELP reduces osteoclast number and activity in ovariectomized mice, underlying its physiological and/or pathological importance in skeletal remodeling.