Tank-binding kinase 1 (TBK1) serves as an important component of multiple signaling pathways. While the majority of research on TBK1 has focused on its role in innate immunity, critical functions for TBK1 in autophagy and cancer are beginning to emerge. This review highlights recent structural and biochemical studies that provide insights into the molecular mechanism of TBK1 activation and summarizes what is known to date about TBK1 substrate selection. Growing evidence suggests that both processes rely on TBK1 subcellular localization, with a variety of adaptor proteins each directing TBK1 to discrete signaling complexes for different cellular responses. Further study of TBK1-mediated pathways will require careful consideration of TBK1 mechanisms of activation and specificity for proper dissection of these distinct signaling cascades.