Many studies have been conducted to improve liposome's potency towards cancer treatment. Recently, modifications of liposomal formulations turned towards either dual loading of anticancer agents to minimize the dose of each agent or the use of targeting moieties to target them to or near cancer cells for minimizing side effects with maximum treatment. In this study, a natural molecule, calcitriol, was used to improve the effectiveness of doxorubicin loaded liposomes by co-loading approach. Calcitriol treatment alone was reported to create an anti-proliferative effect on several carcinoma cell lines. However, to create an antiproliferative effect, a high dose of calcitriol needs to be used which might result in hypercalcemia. Therefore, co-loading calcitriol into liposomes will improve its efficiency on cells besides increasing hydrophobic calcitriol's chemical stability in circulation. At first, possible doxorubicin cytotoxicity improvement against Namalwa cells by calcitriol pretreatment was investigated. The enhancing effect created was over 40% after 72 hours of calcitriol pretreatment. Upon co-loading into liposomes, enhanced and early cytotoxicity was observed even after 24 hours. Hence, the synergistic effect of calcitriol and doxorubicin on Namalwa cells was shown both in free and liposomal forms for the first time. A confocal study confirmed that early cytotoxicity was related with cell internalization. This potency ended when liposomes targeted non-internalizing antigen, CD20. Liposome co-loading of calcitriol and doxorubicin increases the effectiveness of their synergistic activity by creating a dual delivery system. Liposomal co-loaded calcitriol delivery would overcome calcitriol dose limitation problems and decrease possible side effects of both therapeutic agents.