Abstract: Optimization of process conditions for biosynthesis of Pinoresinol (Pin), Pinoresinol-4-O-β-D-glucopyranoside (PMG) and Pinoresinol diglucoside (PDG) by Phomopsis sp. XP-8 assisted Eucommia ulmoides Oliv. was performed. The effects of the cultured method, the addition of Eucommia ulmoides Oliv. and the time of resting on the production of Pin, PMG and PDG by Phomopsis sp. XP-8 assisted Eucommia ulmoides Oliv. were studied. The antioxidative, anticancer properties of the related products were also investigated. The results showed that the optimal yield of PMG, PDG and Pin was obtained at the condition with the PDB medium contained 3% Eucommia ulmoides Oliv. with resting treatment for 5 d. Under the optimal condition, the free radical scavenging ability for DPPH and ABTS was better than the metabolite liquid significantly. This indicated that the antioxidative substances were generated during the fermentation process. The hepatocellular carcinoma HepG-2 proliferation was suppressed by the extract solution. The pure product verifications of activity of the standard samples (Pin, PMG and PDG) were performed. It indicated that Pin was identified as the main active ingredient that suppresses HepG-2. However, the suppressed effect of the extract solution on HepG-2 is a synergistic effect.