The osteoblastic expression of RANKL, which is essential for the communication between osteoblastic cells and osteoclastogenic cells, is stimulated by locally acting or circulating osteotropic cytokines and hormones such as PTH and 1,25-(OH)(2)-D-3 during the bone remodeling process. However, mechanisms those control subcellular trafficking events, membrane expression and extracellular secretion of the newly synthesized RANKL are still not well understood. In our previous study, we have found that the deficiency of osteoblastic Arl6ip5 (ADP-ribosylation-like factor 6 interacting protein 5), an endoplasmic reticulum (ER)-localized protein belonging to the prenylated rab-acceptor-family, enhanced osteoclastogenesis by increasing RANKL transcription in an ER stress dependent signaling. Here we found that over-expression of hemagglutinin (HA)-tagged Arl6ip5 in UAMS32 stromal/osteoblastic cells inhibited osteoclastogenesis, decreased the amount of soluble RANKL in culture supernatant and increased RANKL retention in ER. Moreover, Arl6ip5 bound with RANKL and disturbed the RANKL-OPG complex in UAMS-32 cells. Finally, 1 to 36 amino acid deletion on the NH2 lumen terminus of Arl6ip5 impaired the interaction between Arl6ip5 and RANKL, restored the level of soluble RANKL and the osteoclastogenic ability. These findings indicated that Arl6ip5 was an anti-catabolic factor by binding with RANKL and disturbing its subcellular trafficking in osteoblast. (C) 2015 Elsevier Inc. All rights reserved.