Early region 1A (E1A) is the first viral protein produced upon human adenovirus (HAdV) infection. This multifunctional protein transcriptionally activates other HAdV early genes and reprograms gene expression in host cells to support productive infection. E1A functions by interacting with key cellular regulatory proteins through short linear motifs (SLiMs). In this study, the molecular determinants of interaction between E1A and BS69, a cellular repressor that negatively regulates E1A transactivation, were systematically defined by mutagenesis experiments. We found that a minimal sequence comprised of MPNLVPEV, which contains a conserved PXLXP motif and spans residues 112–119 in HAdV-C5 E1A, was necessary and sufficient in binding to the myeloid, Nervy, and DEAF-1 (MYND) domain of BS69. Our study also identified residues P113 and L115 as critical for this interaction. Furthermore, the HAdV-C5 and -A12 E1A proteins from species C and A bound BS69, but those of HAdV-B3, -E4, -D9, -F40, and -G52 from species B, E, D, F, and G, respectively, did not. In addition, BS69 functioned as a repressor of E1A-mediated transactivation, but only for HAdV-C5 and HAdV-A12 E1A. Thus, the PXLXP motif present in a subset of HAdV E1A proteins confers interaction with BS69, which serves as a negative regulator of E1A mediated transcriptional activation.