The kinetics of entry into and exit from the cavity of cucurbit[7]uril (CB7) was studied by the stopped-flow method in water at various temperatures using pharmaceutically important natural isoquinoline alkaloids as guest compounds. The rate constant of the alkaloid-CB7 complex dissociation was separately determined exploiting the very strong competitive binding of the 1-adamantylammonium cation to CB7. The enthalpy and entropy of activation for the release of berberine from CB7 were significantly lower than those found in the case of the other alkaloids, suggesting different dissociation dynamics. CB7 initially moved from the energetically most stable position encompassing the isoquinoline moiety of berberine to the smaller benzodioxole part, which could leave the macrocycle with less structural distortion. Despite the same thermodynamic parameters of berberine and palmatine inclusion, the latter compound was encapsulated in and set free from CB7 much slower due to the more substantial steric hindrance. The most rapid entry into CB7 and the most exothermic binding were found for epiberberine and coptisine, the alkaloids substituted with the less spacious dioxole ring on their isoquinoline moiety.