Tight junction (TJ) is the barrier of epithelial and endothelial cells to maintain paracellular substrate transport and cell polarity. As one of the TJ cytoplasmic adaptor proteins adjacent to cell membrane, zonula occludens (ZO) proteins are responsible for connecting transmembrane TJ proteins and cytoplasmic cytoskeleton, providing a binding platform for transmembrane TJ proteins to maintain the barrier function. In addition to the basic structural function, ZO proteins play important roles in signal regulation such as cell proliferation and motility, the latter including cell migration, invasion and metastasis, to influence embryonic development, tissue homeostasis, damage repair, inflammation, tumorigenesis, and cancer progression. In this review, we will focus on the signal regulating function of ZO proteins in inflammation and tumorigenesis, and discuss the limitations of previous research and future challenges in ZO protein research.