A3A is a single-domain enzyme with the highest catalytic activity among the human APOBEC3 proteins23, a known restriction factor24,25, and also likely contributes to carcinogenesis26. In this study we quantified the ssDNA specificity of A3A, and identified the consensus signature sequence as (T/C)TC(A/G). The dinucleotide sequence preference for A3A, TC, which was previously found through activity assays10,20,21 was confirmed and expanded to a preference for pyrimidine-TC-purine. Surprisingly context matters, in that the background nucleotide sequence impacts binding affinity, with essentially no binding observed for Poly A 1 C (Fig. 1b), while Poly T 1 C binds with 35 ± 2 nM affinity28. Furthermore, the length of the ssDNA in which (T/C)TC(A/G) is imbedded within also modulates affinity (Supplementary Fig. 3). Structural analysis of the two A3A–ssDNA complexes containing two distinct, but suboptimal ssDNA sequences have led us to develop a model with intra-DNA interactions for the molecular mechanism for A3A’s specificity to ssDNA. In contrast to previous results27, which implicate the −2 position as defining specificity, the base at this position observed in both A3A–ssDNA co-crystal structures do not make any specific interactions with the protein. Rather, the hydrogen bonding edge of the −2 base is in close proximity to corresponding edge of +1 base, suggesting possible intra-DNA interactions as being determinants of preference. Our molecular modeling confirmed such interactions could stabilize the U-shaped DNA conformation within the A3A active site, explaining the −2 position specificity.