Remodeling of cellular cytoskeletal structures in circulating tumor cells facilitates metastatic spread. Rearrangements of actin and tubulin in a nonadherent environment such as the vasculature produces microtubule-based protrusions termed microtentacles. Microtentacles are highly dynamic tubulin structures formed on free-floating cells when the physical outward force generated by the microtubule network overcomes the inward contractile force of the actin cortex. When cells enter a free-floating environment, microtentacles form within minutes, along a similar timeline to the rapid initiation of calcium signal transduction after mechanical stimulation. Historically, many studies focus on aberrant calcium-mediated oncogenic signaling pathways through receptor overactivation or overexpression to examine long-term (>24 hours) functions and phenotypes. Thus, the acute cellular response that occurs within seconds to minutes of stimulation is often overlooked in cancer biology. This study directly examines the cytoskeletal response of breast cancer cells to fluctuations in intracellular and extracellular calcium content. With the use of TetherChip technology in tandem with confocal microscopy, live cell time course imaging, and quantitative cell-based assays we have defined the rapid signaling mechanisms that mediated the cytoskeletal changes in detached and suspended breast epithelial and tumor cells. These findings reveal an induction of acute elevations of cytoplasmic calcium in tumorigenic and metastatic breast tumor cells. We observed that cells undergo rapid actomyosin contraction and rearrangement through phosphorylation on myosin light chain 2 and dephosphorylation of cofilin to suppress the microtentacle phenotype and functions. Moreover, rapid fluctuations from physiological to low intracellular and extracellular calcium conditions profoundly affect microtentacles in mammary epithelial and breast tumor cells. Although the probability of cancer cells encountering extreme environmental calcium gradient changes, these cells modulate their cytoplasmic calcium homeostasis through various calcium sensitive upstream regulators. Therefore, examining these specific channels, pumps, and receptors could have interesting implications for cancer cell behaviors in the tumor microenvironment. By understanding the acute molecular responses to calcium-mediated signal transduction, we can better develop and profile calcium modulators to target circulating tumor cells for the eventual application of rapidly profiling patient circulating tumor cells.