Signal transducer and activator of transcription 1 (STAT1) is an important mediator for cytokine signal transduction, particularly IFN-γ. Following IFN-γ stimulation, STAT1 is activated through tyrosine phosphorylation. Little is known about the function and regulation of STAT1 dephosphorylation after activation. We studied the regulation and function of STAT1 dephosphorylation in different types of cells and found that the phosphorylated STAT1 was quickly dephosphorylated in most of epithelial cells. Further studies revealed that the dephosphorylation of STAT1 was regulated by cell shape/adhesion. Actin cytoskeleton and extracellular matrix (ECM) proteins mediated the STAT1 dephosphorylation through the T-cell protein tyrosine phosphatase TCPTP. Inactivation of the dephosphorylation system by cell detachment rendered the cells more sensitive to IFN-γ-induced cell death. Our results revealed a novel mechanism in regulating IFN-γ/STAT1 signaling. This cell adhesion and cell cytoskeleton-dependent STAT1 dephosphorylation system may have a role in IFN-γ-mediated immunosurveillance for cancer cells by inducing anoikis of detached metastatic cancer cells. © 2011 Elsevier Inc.