Gibberella moniliformis is a common disease in C. oleifera, and always results in significant economic losses. Phosphoenolpyruvate carboxykinase is a critical gluconeogenic enzyme and catalyzes the first committed step in the diversion of tricarboxylic acid cycle intermediates toward gluconeogenesis. According to the relative conservation of homologous gene, a bioinformatics strategy was applied to clone phosphoenolpyruvate carboxykinase gene of Gibberella moniliformis by blasting search of EST database with homologous gene cDNA of Neurospora crassa and identified. Some characters of the PEPCK that can encode amino acid were analyzed and predicted by the tools of bioinformatics in the following aspects, including the composition of amino acid sequences, physical and chemical, O-glycosylation site and tertiary structure of protein and function. These results showed that the full-length of PEPCK was 1962 bp and it contained a complete ORF (1671bp), encoded 556 amino acids, which are much conserved in ascomycetes. The calculated molecular weight of PEPCK was 61893.1 Da, theoretical pI of 5.77, 20 a-helix, 37 sheets, 7 glycosylation sites; it was a stable protein with active site ATP -binding site, metal-binding site and substrate-binding site.