Phomopsis sp. XP-8, an endophytic fungus from the bark of Tu-Chung (Eucommia ulmoides Oliv) showed capability to biosynthesize pinoresinol (Pin) and pinoresinol diglucoside (PDG) from glucose (glu) and phenylalanine (Phe). To verify the mass flow in the biosynthesis pathway, [13C6]-labeled glu and [13C6]-labeled Phe were separately fed to the strain as sole substrates and [13C6]-labeled products were detected by ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry. As results, [13C6]-labeled Phe was incorporated into [13C6]-cinnamylic acid (Ca) and p-coumaric acid (p-Co), and [13C12]-labeled Pin, which revealed that the Pin benzene ring came from Phe via the phenylpropane pathway. [13C6]-Labeled Ca and p-Co, [13C12]-labeled Pin, [13C18]-labeled pinoresinol monoglucoside (PMG), and [13C18]-labeled PDG products were found when [13C6]-labeled glu was used, demonstrating that the benzene ring and glucoside of PDG originated from glu. It was also determined that PMG was not the direct precursor of PDG in the biosynthetic pathway. The study identified the occurrence of phenylalanine- lignan biosynthesis pathway in fungi at the level of mass flow.