Protamines, arginine-rich DNA-binding proteins, are responsible for chromatin compaction in sperm cells, but their DNA groove preference, major or minor, is not clearly identified. We herein study the DNA groove preference of a short protamine-like cationic peptide before and after phosphorylation, using all-atom molecular dynamics and umbrella sampling simulations. According to various thermodynamic and structural analyses, a peptide in its non-phosphorylated native state prefers the minor groove over the major groove, but phosphorylation of the peptide bound to the minor groove not only reduces its binding affinity but also brings a serious deformation of the minor groove, eliminating the minor-groove preference. As protamines are heavily phosphorylated before binding to DNA, we expect that the structurally disordered phosphorylated protamines would prefer major grooves to enter into DNA during spermatogenesis.