In this paper, we report the lipoplex-mediated deintercalation of anticancer drug doxorubicin (DOX) from the DOX DNA complex under controlled experimental conditions. We used three zwitterionic liposomes, namely, 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), which are widely different in their phase transition temperatures to form a lipoplex with calf thymus DNA in the presence of Ca2+ ions. The study revealed that DPPC being in sol gel phase was more effective in releasing the drug from the DOX-DNA complex compared with liposomes that remain in liquid crystalline phase (DMPC and POPC). The higher extent of drug release in the case of DPPC liposomes was attributed to the stronger lipoplex formation with DNA as compared with that of other liposomes. Owing to the relatively smaller head group area, the DPPC liposomes in their sol-gel phase can absorb a larger number of Ca2+ ions and hence offer a strong electrostatic interaction with DNA. This interaction was confirmed by time resolved anisotropy and circular dichroism spectroscopy. Apart from the electrostatic interaction, the possible hydrophobic interaction between the liposomes and DNA was also taken into account for the observed deintercalation. The successful uptake of drug molecules by liposomes from the drug DNA complex in the post-release period was also confirmed using confocal laser scanning microscopy (CLSM).