Objective: To evaluate the expression of receptor activator of nuclear factor-kappa ligand (RANKL), osteoprotegerin (OPG), osteopontin (OPN), interleukin 1b (IL-1b), and transforming growth factor ß1 (TGF-ß1) in the gingival crevicular fluid (GCF) of teeth subjected to orthodontic forces released by aligners. Materials and Methods: A total of 10 healthy, adult patients were selected to participate in this split-mouth study. The treatment plan was designed to obtain only one movement with the first aligner: distalization of a second molar. GCF samples were obtained from pressure and tension sites of the test tooth and from the mesiobuccal and distobuccal sites of the control tooth. The GCF sample volumes were measured using a Periotron 8000. Levels of TGF-b, IL-1b, RANKL, OPG, and OPN were measured by enzyme-linked immunosorbent assay. Results: IL-1b showed a significant increase at the pressure sites after 1 week and 3 weeks with respect to baseline (P , .05) compared with control sites. The kinetics of TGF-1b and OPN were characterized by a significant increase at the tension sites of the test teeth (P , .05) after 3 weeks from the application of orthodontic force. The RANKL level was significantly increased at pressure and tension sites after 1 hour and after 1 week from the application of the orthodontic forces (P ¼ .023 and P ¼ .043, respectively). Conclusions: An increased concentration of bone modeling and remodeling mediators at the pressure sites (IL-1b, RANKL) and tension sites (TGF-1b, OPN) was observed. These scenarios are compatible with previous in vivo and in vitro studies investigating the biological effects of orthodontic tooth movement. (Angle Orthod. 2017;87:74-81)