In this work, anion-π interactions between sulfate groups (SO 4 2-) and protein aromatic amino acids (AAs) (histidine protonated (HisP), histidine neutral (HisN), tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe)) in an aqueous environment have been analyzed using quantum chemical (QC) calculations and molecular dynamics (MD) simulations. Sulfates can occur naturally in solution and can be contained in biomolecules playing relevant roles in their biological function. In particular, the presence of sulfate groups in glycosaminoglycans such as heparin and heparan sulfate has been shown to be relevant for protein and cellular communication and, consequently, for tissue regeneration. Therefore, anion-π interactions between sulfate groups and aromatic residues represent a relevant aspect to investigate. QC results show that such an anion-π mode of interaction between SO 4 2- and aromatic AAs is only possible in the presence of water molecules, in the absence of any other cooperative non-covalent interactions. Protonated histidine stands out in terms of its enhancement in the magnitude of interaction strength on solvation. Other AAs such as non-protonated histidine, tyrosine, and phenylalanine can stabilize anion-π interactions on solvation, albeit with weak interaction energy. Tryptophan does not exhibit any anion-π mode of interaction with SO 4 2-. The order of magnitude of the interaction of aromatic AAs with SO 4 2- on microsolvation is HisP > HisN > Tyr > Trp > Phe. Atoms in molecules (AIM) analysis illustrates the significance of water molecules in stabilizing the divalent SO 4 2- anion over the π surface of the aromatic AAs. MD simulation analysis shows that the order of magnitude of the interaction of SO 4 2- with aromatic AAs in macroscopic solvation is HisP > HisN, Tyr, Trp > Phe, which is very much in line with the QC results. Spatial distribution function analysis illustrates that protonated histidine alone is capable of establishing the anion-π interaction with SO 4 2- in the solution phase. This study sheds light on the understanding of anion-π interactions between SO 4 2- and aromatic AAs such as His and Tyr observed in protein crystal structures and the significance of water molecules in stabilizing such interactions, which is not feasible otherwise.