The nucleus and mitochondria are on correlative dependence; they interact in the process of protein transportation and energy metabolism. The compatibility of nucleus and mitochondria is essential for interspecies somatic cell nuclear transfer (iSCNT) and xenomitochondrial cybrid. In order to test the compatibility of nucleus and mitochondria among human, mouse, and pig cells, we compared the performances of cybrids that fused inter- and intra-species. The ρ0 cells from human and pig cell lines were created as nucleus donors which were transfected with GFP-neo for cell selective system in advance, and mitochondria donor cells were labeled by Mitochondria-RFP. Human and mouse platelets were also used as a mitochondrial donor. Results indicated that all interspecies cybrids declined to die in 2-4 d after the cell fusion in the selection medium, while intraspecies cybrid cells survived and formed stable clones. As a conclusion, the incompatibility between nucleus and mitochondria is the critical factor for the formation of interspecies cybrids.