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O‐linked N‐acetylglucosamine (O‐GlcNAcylation) is a ubiquitous post‐translational modification of proteins that is essential for cell function. Perturbation of O‐GlcNAcylation leads to altered cell‐cycle progression and DNA damage response. However, the underlying mechanisms are poorly understood. Here, we develop a highly sensitive one‐step enzymatic strategy for capture and profiling O‐GlcNAcylated proteins in cells. Using this strategy, we discover that flap endonuclease 1 (FEN1), an essential enzyme in DNA synthesis, is a novel substrate for O‐GlcNAcylation. FEN1 O‐GlcNAcylation is dynamically regulated during the cell cycle. O‐GlcNAcylation at the serine 352 of FEN1 disrupts its interaction with Proliferating Cell Nuclear Antigen (PCNA) at the replication foci, and leads to altered cell cycle, defects in DNA replication, accumulation of DNA damage, and enhanced sensitivity to DNA damage agents. Thus, our study provides a sensitive method for profiling O‐GlcNAcylated proteins, and reveals an unknown mechanism of O‐GlcNAcylation in regulating cell cycle progression and DNA damage response. [ABSTRACT FROM AUTHOR]