Myriad applications of surfactin in environmental and biomedical field prompt understanding the self-assembly behaviour of surfactin in aqueous solution as well as its interaction with counterions. Effect of four divalent counterions namely, Ni(2+), Zn(2+), Cd(2+), and Ca(2+) on the self-assembly of the surfactin, a biosurfactant isolated from Bacillus subtilis YB7 is studied by fluorescence spectroscopy, dynamic light scattering, optical and electron microscopic studies. The critical micelle concentration (CMC) and aggregation number (Nagg) of surfactin are 96.76 ± 15.49 μM and 101.12 ± 2.53, respectively. The degree of counterion association increases as its ionic radius decreases. Ni(2+) exhibits the highest and Ca(2+) the least degree of counterion association. Addition of counterion reduces the size of the microstructures, aggregation number (Nagg) and zeta potential. The reduction in the zeta potential indicates the neutralization of the negative charges on the electrical double layer of the microstructures. Differential interference contrast (DIC) and transmission electron microscopic (TEM) images of surfactin show the presence of vesicles and large aggregates including giant vesicles. On the addition of Ca(2+), fusion of vesicles into large aggregates is predominantly observed. Ni(2+) induces the transition of large spherical vesicles into small spherical, worm-like vesicles and multicompartment-like structures (vesosome). Such structures are the evidences for metal ion coordinated intervesicular interactions. This study reveals that the self-assembly process of surfactin can be controlled by the addition of metal ions according to the requirements.