The invention discloses Pichia pastoris strain with alpha-1, 6-mannosyl transferase absent and the establishing method, which belongs to the biological engineering field. The collection number of the Pichia pastoris strain with alpha-1, 6-mannosyl transferase absent is CGMCC No.1853. The invention has the advantages that the Pichia pastoris strain with alpha-1, 6-mannosyl transferase absent built by the invention can prevent the generation of excessive manna saccharify, when expressing extrinsic glucoprotein, reduce the immunogenicity of the pressed protein, and therefore, the invention has important application value in the biomedical field, and other fields; simultaneously, the strain can also be applied to the further gene knockout or the metabolic engineering reconstruction. The invention also builds a method for knocking out the alpha-1, 6-mannosyl transferase gene through secondary homologous recombination, mutant strain can be relatively easily obtained through the method, therefore the sieving workload is greatly reduced, and the success ratio is improved.