Metastatic tumors are the primary cause of death in patients with breast cancer. Recent data indicate that the peroxisome proliferator-activated receptor γ (PPARγ) ligands, thiazolidinediones (TZDs), possess anti-invasive activities on human breast cancer cells. However, the effects of TZDs on other metastatic properties of breast cancer cells such as adhesion, spreading, and migration are not well established. In this study, we show that troglitazone (TG), a member of the TZD family, inhibits lamellipodia formation or membrane ruffling as well as actin polymerization at these structures in MDA-MB-231 and T47D breast cancer cells. In addition, TG reduces migration, adhesion, and spreading on fibronectin (FN)-coated plates. These phenomena were associated with the dramatic decrease of Tyr397 and Tyr576 phosphorylation of focal adhesion kinase (FAK) and the detergent-insoluble Rac1. We also found that TG upregulates Tyr416 phosphorylation of Src, but downregulates the Src–FAK complex. Moreover, we use a PPARγ-inactive derivative of TG (STG28) and a PPARγ antagonist (GW9662) to eliminate PPARγ-mediated effects. We found that treatment with STG28 or GW9662 plus TG showed similar effects compared to TG treatment alone on tyrosine phosphorylation of FAK and Src, indicating that these effects are not the result of PPARγ activation. Interestingly, we found that TG upregulates actin filament assembly at the point of cell–cell contact in T47D cells, indicating that TG may also upregulate cell–cell adhesion in breast cancer cells which express E-cadherin. These results suggested that TG should be investigated further for its therapeutic potential in metastatic breast cancer. © 2008 Wiley-Liss, Inc.