The report of the existence of 5-hydroxymethylcytosine (hm5C) in mammalian genomes is a milestone discovery. hm5C is now generally viewed as the sixth base of DNA with important functions on epigenetic regulation. The in-depth investigation of the biological functions of hm5C requires elucidating the distribution patterns of hm5C in genomes, better in single-nucleotide resolution. It was reported that the cytosine deaminases of the APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide-like) family are nucleic acid editing enzymes and can deaminate cytosine (C) to form uracil (U). Particularly, a subfamily of APOBEC (APOBEC3A) can efficiently deaminate both C and 5-methylcytosine (m5C). In the current study, we identified that APOBEC3A protein can effectively deaminate C, m5C, and hm5C but shows no observable deamination activity toward glycosylated hm5C (β-glucosyl-5-hydroxymethyl-2'-deoxycytidine, ghm5C) by using the restriction enzyme-based assay and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. By virtue of the differential deamination activity of APOBEC3A toward C, m5C, and ghm5C in conjugation with sequencing, we developed the single-nucleotide resolution analysis of hm5C in DNA. In this analytical strategy, the original C and m5C in DNA will be deaminated by APOBEC3A to form U and thymine (T), both of which will read as T during sequencing, while ghm5C is resistant to deamination and will read as C during sequencing. Therefore, the remaining C in the sequence context only could come from original hm5C, which offers the single-nucleotide resolution analysis of hm5C in DNA. This APOBEC3A-mediated deamination sequencing (AMD-seq) is straightforward and involves no bisulfite treatment, which avoids the substantial degradation of DNA. Future application of this strategy can be performed for the reliable mapping of hm5C in genome-wide scale at the single-nucleotide resolution.