Intact insulin signaling and glucose transport in adipocytes are crucial to maintaining whole-body energy metabolism. Focal adhesion kinase stands as a central intracellular protein facilitating signaling between the extracellular matrix and the cytoplasm, thereby regulating cellular metabolism. Here, we have investigated the role of focal adhesion kinase in adipocyte glucose transport using an array of methods, including affinity purification combined with quantitative mass spectrometry, glucose tracer assays, western blotting, and confocal imaging. Pharmacological inhibition (PF-573228) of focal adhesion kinase suppressed the interaction of focal adhesion kinase with numerous actin-associated proteins, reduced Rac1 activity, as well as phosphorylation of the Rac1 downstream target PAK1/2, and further led to impaired GLUT4 translocation and glucose uptake. In summary, we demonstrate that focal adhesion kinase plays a key role in controlling actin remodeling, subsequent GLUT4 translocation, and ultimately glucose transport in adipocytes.