Cathepsin K (CTSK) is a secreted protease that plays an essential role in osteoclastic bone resorption, and CTSK levels increase with osteoclast differentiation and activation, a process that is controlled by a complex physiological network of hormones and cytokines. A critical regulator of this process is receptor activator of NF-kappaB ligand (RANKL), a member of the tumor necrosis factor (TNF) superfamily of cytokines that can act via the TNF receptor activating factor (TRAF6)/AP-1 signaling pathway. However, the mechanism whereby RANKL modulates CTSK expression is not fully understood. Therefore, we investigated the regulation of CTSK expression and promoter activity in RAW 264.7 osteoclast precursor cells, which can be readily differentiated to osteoclasts upon RANKL stimulation. Western blot analysis, real-time RT-PCR and luciferase reporter gene assays revealed that RANKL stimulated CTSK expression and promoter activity in a dose- and time-dependent manner and that this activation was inhibited by either dominant negative (DN) TRAF6 or DN-c-fos. TRAF6 stimulated the basal activity of a truncated CTSK promoter, and DN-c-fos blocked this stimulation. JunB alone also stimulated basal CTSK promoter activity, whereas c-jun, JunD or c-fos alone did not. However, co-transfection of any of these jun-family members with c-fos (AP-1) significantly increased CTSK promoter expression. siRNA targeted against c-jun or junB suppressed RANKL-mediated CTSK expression. Therefore, both TRAF6 and AP-1 help regulate the basal and RANKL-mediated stimulation of CTSK gene expression in RAW 264.7 cells.