The relaxation activity of topoisomerase I is required for regulation of global and local DNA supercoiling. The in vivo topoisomerase I enzyme activity is sensitive to lysine acetylation⁻deacetylation and can affect DNA supercoiling and growth as a result. Nonenzymatic lysine acetylation by acetyl phosphate has been shown to reduce the relaxation activity of topoisomerase I. In this work, the biochemical consequence of topoisomerase I modification by acetyl phosphate with enzymatic assays was studied. Results showed that noncovalent binding to DNA and DNA cleavage by the enzyme were reduced as a result of the acetylation, with greater effect on DNA cleavage. Four lysine acetylation sites were identified using bottom-up proteomics: Lys13, Lys45, Lys346, and Lys488. The Lys13 residue modified by acetyl phosphate has not been reported previously as a lysine acetylation site for topoisomerase I. We discuss the potential biochemical consequence of lysine acetylation at this strictly conserved lysine and other lysine residues on the enzyme based on available genetic and structural information.