All-trans-retinoic acid (ATRA) shows anti-cancer activities, especially in patients with acute promyelocytic leukemia. Due to the highly variable bioavailability of ATRA and induction of its own metabolism after oral treatment, development of alternative parenteral dosage form is required. The principal aim of this study was to develop a parenteral formulation of ATRA by overcoming its solubility limitation by utilizing phospholipid-based microemulsion system as a carrier. Microemulsion was prepared with pharmaceutically acceptable ingredients such as soybean oil and phospholipids. The mean particle diameter and polydispersity of ATRA microemulsion could be decreased to be applicable for parenteral administration by modulation of composition of microemulsion. The loading concentration of ATRA in microemulsion increased by increasing the oil contents and also by inclusion of distearoylphosphatidyl-ethanolamine-N-poly(ethyleneglycol) 2000 (DSPE-PEG). Furthermore, loading of ATRA in microemulsion improved the chemical stability of ATRA. The pharmacokinetic profile of ATRA after intravenous injection of microemulsion formulation to rats was similar to that of sodium ATRA. The growth inhibitory effects of ATRA on human cancer HL-60 and MCF-7 cell lines were also similar between free ATRA and microemulsion formulation of ATRA, suggesting that its anti-cancer activity was not impaired by loading in microemulsion. Our study herein demonstrates that phospholipid-based microemulsion may provide an alternative parenteral formulation of ATRA.