Protein insertion into membranes is a process occurring in every cell and every cellular compartment. Yet, many thermodynamic aspects of this fundamental biophysical process are not well understood. We investigated physicochemical parameters that influence protein insertion using the model protein KcsA, a 2-transmembrane ion channel. To understand what drives insertion and to identify individual steps of protein integration into a highly apolar environment, we investigated the contribution of electrostatic interactions and lipid composition on protein insertion on a single molecule level. We show that insertion of KcsA is spontaneous and directional as the cytosolic part of the protein does not translocate across the membrane barrier. Surprisingly, not hydrophobic residues but charged amino acids are crucial for the insertion of the unfolded protein into the membrane. Our results demonstrate the importance of electrostatic interactions between membrane and protein during the insertion process of hydrophobic polypeptides into the apolar membrane. On the basis of the observation that negatively charged lipids increase insertion events while high ionic strength in the surrounding aqueous phase decreases insertion events, a two-step mechanism is proposed. Here, an initial electrostatic attraction between membrane and protein represents the first step prior to insertion of hydrophobic residues into the hydrocarbon core of the membrane.