During invasion, cancer cells communicate with the surrounding environment through dynamic and bidirectional interactions. Based on the paracrine interactions, cancer cells acquire invasive properties by changing their morphology and mode of migration. Thus, inhibition of the morphological change induced by the paracrine interactions would be served as promising candidates for anticancer therapies. In this study, we demonstrate that timosaponin A-III (TAIII), a steroidal saponin isolated from the roots of Anemarrhena asphodeloides, disturbs morphological plasticity and migratory activity of breast adenocarcinoma cells based on the paracrine interaction with mammary epithelium-derived cells. TAIII suppressed lamellipodia formation in MDA-MB-231 cells induced by conditioned medium from MCF10A, thereby inhibiting morphological changes and migration. TAIII also inhibited membrane spreading induced by overexpression of constitutively active form of Rac1 in HeLa cells, inducing cell contraction and expanded intercellular gaps. Interestingly, the activity of TAIII against membrane dynamics was unique among steroidal saponins and their aglycones contained in Anemarrhena asphodeloides. We also found that TAIII clearly blocked internalisation of integrin complex, so that TAIII-treated cells failed to attach and spread onto extracellular matrix. These results provide a novel explanation how TAIII inhibits cancer cell migration and support a possibility of TAIII as a candidate for cancer therapeutic agent.