In this study, we evaluated changes in focal adhesions (FAs) in two types of breast cancer cell (BCC) lines (differentiated MCF-7 and the triple-negative MDA-MB-231 cell line) exposed to simulated microgravity (s-μ) created by a random positioning machine (RPM) for 24 h. After exposure, the BCC changed their growth behavior and exhibited two phenotypes in RPM samples: one portion of the cells grew as a normal two-dimensional monolayer [adherent (AD) BCC], while the other portion formed three-dimensional (3D) multicellular spheroids (MCS). After 1 h and 30 min (MDA-MB-231) and 1 h 40 min (MCF-7), the MCS adhered completely to the slide flask bottom. After 2 h, MDA-MB-231 MCS cells started to migrate, and after 6 h, a large number of the cells had left the MCS and continued to grow in a scattered pattern, whereas MCF-7 cells were growing as a confluent monolayer after 6 h and 24 h. We investigated the genes associated with the cytoskeleton, the extracellular matrix and FAs. , and gene expression patterns were not significantly changed in MDA-MB-231 cells, but we observed a down-regulation of mRNAs in AD cells and of and mRNAs in MDA-MB-231 MCS. RPM-exposed MCF-7 cells revealed a down-regulation in the gene expression of and in AD cells and and in MCS. An interaction analysis of the examined genes involved in 3D growth and adhesion indicated a central role of fibronectin, vinculin, and E-cadherin. Live cell imaging of eGFP-vinculin in MCF-7 cells confirmed these findings. β-catenin-transfected MCF-7 cells revealed a nuclear expression in 1 and RPM-AD cells. The target genes and of the Wnt/β-catenin signaling pathway were differentially expressed in RPM-exposed MCF-7 cells. These findings suggest that vinculin and β-catenin are key mediators of BCC to form MCS during 24 h of RPM-exposure.