A uni-molecular layer of lipids at air-water interface mimicking one of the leaflets of the cellular membrane provides a simple model to understand the interaction of any foreign molecules with the membrane. Here, the interactions of protein Kalata B1 (KB1) of cyclotide family with the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG), and 1,2-distearoyl-sn-glycero-3-ethylphosphocholine chloride salt (DSEPC) have been investigated. The addition of KB1 induces a change in pressure of the lipid monolayers. The characteristic time of the change in pressure is found to be dependent on the electrostatic nature of the lipid. Even though the protein is weakly surface active, it is capable of modifying the phase behavior and elastic properties of lipid monolayers with differences in their strength and nature making the layers more floppy. The KB1-lipid interaction has been quantified by calculating the excess Gibb's free energy of interaction and the 1-anilino-8-naphthalenesulfonate (ANS) binding studies. The interaction with zwitterionic DPPC and negatively charged DPPG lipids are found to be thermodynamically favorable whereas the protein shows a weaker response to positively charged DSEPC lipid. Therefore, the long ranged electrostatic is the initial driving force for the KB1 to recognize and subsequently attach to a cellular membrane. Thereafter, the hydrophobic region of the protein may penetrate into the hydrophobic core of the membrane via specific amino acid residues. © 2021 Elsevier B.V.