[Objective] The purpose was to clone and express endo-1,4-β-glucanase gene of Humicola insolens in Pichia pastoris expression system. [Methods] An endo-1,4-β-glucanaseⅡ(egⅡ) cDNA gene was isolated from the fungus Humicola insolens NC3 by RT-PCR. Subsequently, we cloned the egⅡgene into expression vector pPIC9K, and then transformed into Pichia pastoris GS115. [Results] SDS-PAGE and CMC enzyme activity analysis demonstrated that recombinant EGⅡ protein was successfully expressed after induction in shake flasks. The endo-1,4-β-glucanase exhibited maximum activity at 70 °C and pH 6.5, and was stable between pH 6.0 and 7.0 and below 65 °C. [Conclusion] P. pastoris expression system is an efficient way of production of endo-1,4-β-glucanase. The recombinant endo-1,4-β-glucanase could be a candidate for industrial applications.