Carbonic anhydrase (CA) is an important enzyme, which has multiple isoforms each performing different functions in humans. Characterizing the interactions of carbonic anhydrase isoforms with ligands is a difficult but important task. In recent years, steered molecular dynamics (SMD) has been used successfully to characterize ligand binding and unbinding events. In this study, constant velocity SMD simulations were performed on four cytosolic human carbonic anhydrases (carbonic anhydrase I, II, III and VII) using a ligand called Coumarin2 5Z/5E to understand better the nature of interactions between this ligand and the various enzyme isoforms. The influence of force constant and pulling velocity on SMD simulations were investigated. When the force constant and the pulling velocity were changed, no significant effect on interaction profiles was observed. Finally, hydrogen bond interactions, Histidine 64 positions and the differences between force-distance profiles for all isozymes were analyzed. These results demonstrate that human cytosolic carbonic anhydrases I, III, and VII show similar Coumarin2 5Z/5E unbinding patterns with different rupture forces, while carbonic anhydrase II has a distinct interaction profile.