Abstract: The recombinant Pichia pastoris GS115/HPV16L1 (mut^s) fermentation conditions for human papillomavirus the virus-like particles (HPV VLPs) were optimized. Based on the defined shaking flask medium, effects of pH of the culture medium, temperature, ammonium sulfate, and oleic acid were investigated during the induction period in human papillomavirus virus-like particles fermentation. The results showed that the monomeric protein L1 expression conditions were remarkably different from the conditions for assembly. During HPV L1 fermentation process, the optimized conditions for L1 expression are those: pH=4. 5, temperature 28 ℃, and ammonium ion concentration 0. 26-0. 30 mol/L at the exponential growth phase. However, for VLPs production, the culture conditions are as follows: pH=3. 5, temperature 30 ℃, and the ammonium ion concentration 0. 34-0. 42 mol/L controlled at the exponential phase. In addition, the monomer L1 expression was inhibited with the addition of oleic acid at the initial induction period. Oleic acid (φ=5%) could promote the monomer L1 self assembly at the middle induction phase. The relationship between the intracellular monomer L1 expression and its self assembly can be applied for the expression of the human papillomavirus virus-like particles by recombinant Pichia pastoris in industrial production.