The upstream stimulatory factors (USFs) are regulators of important cellular processes. Both USF1 and USF2 are supposed to have major roles in metabolism, tissue protection and tumor development. However, the knowledge about the mechanisms that control the function of USFs, in particular in tissue protection and cancer, is limited. Phosphorylation is a versatile tool to regulate protein functions. Thereby, phosphorylation can positively or negatively affect different aspects of transcription factor function including protein stability, protein–protein interaction, cellular localization, or DNA binding. The present review aims to summarize the current knowledge about the regulation of USFs by direct phosphorylation and the consequences for USF functions in tissue protection and cancer.