A series of stable natural diatomite-supported CaO catalysts were synthesized for biodiesel production. All materials have been thoroughly characterized by Thermogravimetric analysis (TGA), Scanning electron microscopy (SEM), Energy dispersive spectrometer (EDS), Fourier transform-infrared spectroscopy (FTIR), IR-Raman, X-ray diffraction (XRD), N-2 adsorption/desorption, and Hammett indicator. The results indicated that 30Ca/Di_800 catalyst exhibited the highest catalytic activity (a biodiesel yield of 92.4% was acquired) owing to its highest total basicity. Meanwhile, the superior stability of the catalyst during reaction was also demonstrated. The catalyst can be recycled 10 times without sighificant loss of catalytic efficiency. Therefore, natural diatomite not only can be successfully used as the support for the synthesis of catalyst but also can strongly interact with CaO to form the stable CaO-Si-bond. Furthermore, the as synthesized catalyst also exhibited good better water-tolerant ability. The Ca-O-Si bond existed on the catalyst surface considered to be a main factor for its high stability and water-tolerance. (C) 2017 Elsevier Ltd. All rights reserved.