In this work we prepared and characterized two liposomal formulations of a semisynthetic nitric oxide (NO)-releasing doxorubicin (Dox), called nitrooxy-Dox (NitDox), which we previously demonstrated to be cytotoxic in Dox-resistant human colon cancer cells. Liposomes with 38.2% (Lip A) and 19.1% (Lip B) cholesterol were synthesized: both formulations had similar size and zeta potential values and caused the same intracellular distribution of free NitDox, but Lip B accumulated and released NitDox more efficiently. In Dox-resistant human colon cancer cells, Lip A and Lip B exhibited a more favorable kinetics of drug uptake and NO release, and a stronger cytotoxicity than Dox and free NitDox. While Caelyx, one of the liposomal Dox formulations approved for breast and ovary tumors treatment, was ineffective in Dox-resistant breast/ovary cancer cells, Lip B, and to a lesser extent Lip A, still exerted a significant cytotoxicity in these cells. This event was accompanied in parallel by a higher release of NO, which caused nitration of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two transporters involved in Dox efflux, and impaired their pump activity. By doing so, the efflux kinetics of Dox after treatment with Lip B was markedly slowed down and the intracellular accumulation of Dox was increased in breast and ovary drug-resistant cells. We propose these liposomal formulations of NitDox as new tools with a specific indication for tumors overexpressing Pgp and MRP1.