Cytarabine (Ara-C) is an attractive chemotherapeutic agent used for the treatment of acute myeloblastic leukemia, however, its severely drawbacks such as low lipophilicity and rapid plasma degradation limit clinical applications. Here, we synthesized a new Ara-C prodrug DTA-Ara by conjugating 2-decyltetradecanoic acid (DTA), a double-chained fatty acid with 24 carbons with Ara-C. It was the first time to see that DTA-Ara molecules could self-assemble into stable spherical nanoparticles (NPs) in aqueous solution with extremely high drug loading (63wt%). The DTA-Ara NPs had the average sizes of approximately 130nm and a zeta potential around −31.6mV. Importantly, the DTA-Ara NPs were stable in deionized water or phosphate buffer solution (PBS, pH7.4) solution for more than one week and the hemolysis rate was <10%, which indicated that it could be administrated intravenously. Moreover, the in vitro cytotoxicity study further manifested that the resulting prodrug showed the marked antitumor activity against human leukemia cell line K562 and HL60 cells compared to the naked drug ascribed to its improvement of the hydrophobicity and biomembrane penetrability. The strategy of delivering lipophilized nucleoside analogs using prodrug self-assembling nanoparticles demonstrate potential superiority for Ara-C and provide a new promising therapeutic schedule for leukemia.