Zinc oxide (ZnO) nanoparticles were synthesized by a reverse microemulsion system formed from sodium bis(2-ethylhexyl)sulfosuccinate (Aerosol OT, or AOT):glycerol:n-heptane. The zinc precursor was zinc acetate dihydrate. The formation of ZnO nanoparticles was achieved by calcination of premature zinc glycerolate microemulsion product in air at 300, 400 and 500 °C. The crystal structure and the morphology of the ZnO nanoparticles were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Thermal analysis was employed to reveal structural and chemical changes during calcination. Both surfactant concentrations – AOT – in the initial microemulsion formulation and the calcination temperature influenced the morphology and size of the ZnO nanoparticles. Low surfactant concentrations (5:5:90, AOT:glycerol:n-heptane, wt.%) resulted in formation of spherical ZnO nanoparticles. The average particle size increased from 15 ± 1 to 24 ± 1 nm with calcination temperature, but spherical morphology remained unchanged after all calcination treatments. The microemulsion system containing higher surfactant amount (30:5:65, AOT:glycerol:n-heptane, wt.%) resulted in rod-like ZnO nanostructures after calcination at 300 and 400 °C, with a diameter of 22 ± 3 and 28 ± 1 nm; and with a length of 66 ± 3 and 72 ± 1 nm, respectively. Further increase in the calcination temperature to 500 °C initiated rod-to-sphere shape transformation for the ZnO nanoparticles produced using this particular microemulsion formulation. For all ZnO microemulsion products, the photoluminescence measurements suggested a high defect concentration which increases with calcination temperature.