Epidermal growth factor (EGF) is a therapeutically important member of a family of the growth factors that activate EGF receptors, triggering several signaling pathways vital for development, homeostasis and regeneration of many organs. Previous studies have shown the ability of S100A4, a small calcium-binding protein of the S100 family, to directly affect functional activity of EGF receptors and its ligands, including EGF. However, selectivity and functional significance of EGF-S100 interactions remain unexplored. To address this, we examined specificity of EGF for twenty-one S100 proteins using surface plasmon resonance spectroscopy. The impact of the revealed interactions on EGF-induced cellular effects was assessed using cell proliferation and metabolic activity assays. S100A2/A4/A6/A11/A12/A13/A16/P and S100A8-S100A9 heterodimer bind EGF strictly in the presence of calcium with lowest estimates of the equilibrium dissociation constant ranging from 13 nM to 1.0 μM. Structural modelling indicates involvement of α-helix IV and "hinge" region of the EGF-specific S100 proteins in the EGF binding, which was confirmed for S100P by mutagenesis. The complexation of EGF with S100A2/A6/A12/A16/P exerts distinct S100-dependent effects on viability and metabolic activity of lung adenocarcinoma A549 cells. Bioinformatics analysis showed that dysregulation of EGF and the EGF-specific S100 proteins is associated with many oncological diseases, Alzheimer's and Parkinson's diseases, psoriasis, etc. Overall, specific S100 proteins have been shown to directly modulate various aspects of EGF functioning, which may be important for ensuring efficacy of the EGFR-targeted cancer therapies, as well as for use of EGF in regenerative medicine.