Golgi fragmentation and ER exit site disassembly are considered to be the leading causes of the mitotic block of secretion from the ER. Although the mechanisms of Golgi fragmentation have been extensively characterized, ER exit block early in mitosis is not well understood. We previously reported that TANGO1 organizes ER exit sites by directly interacting with Sec16. We identified TANGO1 as a regulator of ER exit site disassembly during mitosis. TANGO1 phosphorylation was coordinately regulated by a kinase (CK1) and a phosphatase (PP1). CK1-mediated TANGO1 phosphorylation reduces binding to Sec16, leading to the disassembly of ER exit sites. CK1 constantly phosphorylates TANGO1, whereas PP1-mediated dephosphorylation of TANGO1 decreases during mitosis. Thus, the phosphorylation status of TANGO1, which is controlled by balanced activities of the kinase CK1 and the phosphatase PP1, regulates the organization of ER exit sites during the cell cycle.