The metastasis occurrence, an important prognostic factor, depends on peculiarities such as cellular invasiveness and cell migration, mechanisms controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). An increased expression of this protein promotes tumor growth and metastasis, a mechanism which can be restricted by the use of the effector9s inhibitors. Melatonin, a hormone secreted by the pineal gland, has shown oncostatic action and anti-metastatic effects and can modulate the ROCK-1expression. The objective of this study was to investigate the anti-metastatic response mediated by ROCK-1 and through the action of melatonin and its specific inhibitor (Y27632) in vitro and in vivobreast cancer models. Cells from metastatic (MDA-MB-231) and non-metastatic (MCF-7) breast cancer lines were treated with melatonin and Y27632. Cell viability was verified by MTT assay, cell migration/invasion assays in Boyden chamber, ROCK-1 protein and gene expression by western blot and quantitative real time PCR, respectively. In addition, the in vivometastasis study was performed using female athymic nude mice induced by injection of 2x105 MDA-MB-231 viable cells by tail veinfor lung metastasis and byintracardiac for bone metastasis, during 3 weeks. The animals were treated with melatonin and Y27632 for 2 weeks. The metastasis developments were evaluated by single photon emission computed tomography (SPECT). Treatment with melatonin reduced cell viability and migration of both cell lines (p 0.05). An statistically significant reduction of ROCK-1 gene expression was observed in all treatment groups (p Support: FAPESP. Citation Format: Debora C Zuccari, Thaiz F Borin, Ali S Arbab, Livia C Ferreira, Marina G Moschetta, Gustavo R Martins, Larissa B Maschio, Vanessa A Fabri, Verena B Coimbra. Melatonin9s inhibitory effect on breast cancer metastasis mediated by ROCK-1 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-20.