Objective To transfect EGFP gene to porcine embryonic fibroblasts (PEFs) of Tibetan miniature pigs by Lonza Nucleofector II machine and compare the tansfection efficiency between this method and the lipofection method. Method A plasmid carrying green fluorescent protein (GFP) was transfected into PEFs of Tibetan miniature pigs via the Lonza Nucleofector II machine (program U020) and by Lipofectamine 2000. Results 5 hours after nucleofection, green fluorescence was observed, indicating 80% transfecting efficiency in the nucleofection group, which is significantly higher than the lipofection group. Conclusion Nucleofector II machine can efficiently transfect PEFs, provides a reliable method for efficiently generate transgenic Tibetan minipigs.