This paper investigates the correlations between interfacial reaction, crystallographic orientation relationship on the interface and the required undercooling for nucleation on different crystallographic planes of MgO. Thermal analysis and high resolution transmission electron microscopy were used to study the nucleation behavior of liquid, high-purity Al droplet on single crystal MgO substrates using a DSC with an integrated image capture system and a sessile drop apparatus. The results showed that the original substrate MgO would be completely replaced by the reaction product MgAl2O4 at the interface owing to the chemical reaction between liquid Al and the MgO substrates. In addition, the same crystal structure with the original MgO substrate is achieved in the new MgAl2O4 layer. The orientation relationship between MgAl2O4 and Al is consistent with the theoretical prediction according to the Bramfitt's lattice misfit theory and Edge-to-Edge model. Consequently, the generated MgAl2O4 significantly influences the detected undercooling. Display omitted •Only MgAl2O4 layer is produced at the interface of Al/MgO system due to chemical reaction occurred from 750 °C to 660 °C.•The well-defined orientation relationship is achieved at the interface of Al/MgAl2O4/MgO.•MgAl2O4 layer has a significant influence on the heterogeneous nucleation of Al.