The entrapment of fat-soluble vitamins in liposomes is an effective technique to protect them from optical and chemical degradation and oxidation. In this study palmitic acid (PA) was incorporated into the liposomal membrane to increase the stability of the nanoliposomes loaded with vitamin D3 (VD3). The liposomal formulations were prepared by the thin-layer hydration method with different rations of soy phosphatidylcholine (SPC), cholesterol (CHO), and PA as membrane compounds. The encapsulation efficiency (EE%), particle size, particle size distribution, zeta potential, thermal properties, morphology, stability, antioxidant activity, moisture content, solubility, and in vitro release of nanoliposomes were studied. Results showed that the addition of PA to nanoliposomes in the proportions of 1% increased the EE, stability, antioxidant activity, moisture content, and solubility but reduced their particle size, particle size distribution, and zeta potential. Furthermore, the spectra of Fourier transform infrared (FTIR) spectroscopy showed that the complex formed between VD3 and PA was a physical interaction. The high release of VD3 (95.4%) in the intestine medium followed the Fickian diffusion mechanism. Generally, the incorporation of PA in nanoliposomes is a promising approach to improve the stability of VD3-loaded liposomes during storage.