A strong xylophagous activity of termites depends on the symbiotic flagellated protists in the gut; however, few studies have investigated the metabolism of the gut protists because they are very difficult to culture in laboratories. The primary metabolic pathway was reconstructed from meta-EST analysis of the symbiotic protistan community of the termite Coptotermes formosanus, which harbors only three parabasalid species, Pseudotrichonympha grassii (Trichonymphida), Holomastigotoides mirabile (Spirotrichonymphida), and another spirotrichonymphid. They are considered to make energy in hydrogenosome, an anaerobic hydrogen producing organelle. Two pathways from phosphoenolpyruvate (PEP) were identified; one directly converts PEP to pyruvate by pyruvate phosphate dikinase, while the other produces malate. Genes encoding phosphoenolpyruvate carboxykinase (PCK), a key enzyme of the latter pathway, were likely to have been acquired independently from different bacterial groups to the common ancestors of Trichonymphida and Spirotrichonymphida, respectively. Surprisingly, cytosolic PCK of P. grassii and its recombinant enzyme showed higher affinity to PEP than oxalacetate (OAA), while those of H. mirabile did not. We here propose that P. grassii has the OAA-forming-type PCK and uses malate as a carrier of reducing equivalents to hydrogenosomes, and the independent lateral gene transfer of PCK may have caused this differentiation of primary metabolism.