Updated project metadata. CK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe a new mechanism of CK1 regulation conserved from yeast to human – autophosphorylation of a threonine in the mobile L-EF loop proximal to the active site – that inhibits kinase activity. Consequently, yeast and human CK1 enzymes with phosphoablating mutations at this site are hyperactive in vitro. We used quantitative phosphoproteomics to show that disruption of this regulatory mechanism rewires CK1 signaling in Schizosaccharomyces pombe. In accord, we found that a known CK1 pathway, a mitotic checkpoint, is downregulated in these mutants, while new pathways that confer heat shock resistance and suppress meiotic transcripts are upregulated. Molecular dynamics simulations demonstrated that phosphorylation on the L-EF loop alters the conformation of the substrate docking site, and we propose that this affects which CK1 substrates can be phosphorylated. Due to the functional importance of the L-EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo.