Breast cancer metastasis suppressor 1 (BRMS1) is a predominantly nuclear protein that suppresses metastasis in multiple human and murine carcinoma cell lines. BRMS1 interacts with several nuclear proteins including SIN3:HDAC chromatin remodeling complexes that are involved in repressing transcription. However, recent reports suggest BRMS1 may function in the cytoplasm. BRMS1 has two predicted nuclear localization sequences (NLS) that are located near the C-terminus (amino acids 198-205 and 238-244, NLS1 and NLS2 respectively). We hypothesized that nuclear localization sequences of BRMS1 were essential for BRMS1 mediated metastasis suppression. Replacement of NLS2 with NLS1 (BRMS1NLS1,1), truncation at 238 (BRMS1ΔNLS2), or switching the location of NLS1 and NLS2 (BRMS1NLS2,1) did not affect nuclear localization; but, replacement of NLS1 with NLS2 (BRMS1NLS2,2) or truncation at 197 (BRMS1ΔNLS which removes both NLS) promoted cytoplasmic localization. MDA-MB-231 human metastatic breast cancer cells transduced with BRMS1NLS1,1, BRMS1NLS2,2 or BRMS1NLS2,1 were evaluated for metastasis suppression in an experimental xenograft mouse model. Interestingly, while NLS2 was not necessary for nuclear localization, it was found to be important for metastasis suppression since BRMS1NLS2,2 suppressed metastasis by 85%. In contrast, BRMS1NLS2,1 and BRMS1NLS1,1 did not significantly suppress metastasis. Both BRMS1 and BRMS1NLS2,2 co-immunoprecipitated with SIN3A in the nucleus and cytoplasm; however, BRMS1NLS1,1 and BRMS1NLS2,1 were associated with SIN3A in the nucleus only. Moreover, BRMS1 and BRMS1NLS2,2, but not BRMS1NLS1,1 and BRMS1NLS2,1, down-regulated the pro-metastatic microRNA, miR-10b. Together, these data demonstrate an important role for NLS2 in the cytoplasm that is critical for metastasis suppression and is distinct from nuclear localization. © 2013 Hurst et al.