Regulatory factor MBF1 is highly conserved between species and has been described as a cofactor and transcription factor. In plants, several reports associate MBF1 with heat stress response. Nevertheless, the specific physical processes involved in the MBF1–DNA interaction are still far from clearly understood. We thus performed extensive molecular dynamics simulations of DNA with a homology-based modethel of the MBF1 protein. Based on recent experimental data, we proposed two B-DNA sequences, analyzing their interaction with our model of the Arabidopsis MBF1c protein (AtMBF1c) at three different temperatures: 293, 300, and 320 K, maintaining a constant pressure of 1 bar. The simulations suggest that MBF1 binds directly to the DNA, supporting the idea of its role as a transcription factor. We identified two different conformations of the MBF1 protein when bound, and characterized the specific groups of amino acids involved in the formation of the DNA–MBF1 complex. These regions of amino acids are bound mostly to the minor groove of DNA by the attraction of positively charged residues and the negatively charged backbone, but subject to the compatibility of shapes, much in the sense of a lock-and-key mechanism. We found that only with a sequence rich in CTAGA motifs at 300 K does MBF1 bind to DNA in the DNA-binding domain Cro/C1-type HTH predicted. In the rest of the systems tested, we observed non-specific DNA–MBF1 interactions. This study complements findings previously reported by others on the role of CTAGA as a DNA-binding element for MBF1c at a heat stress temperature.