The regulator in glycerol repression of Pichia pastoris AOX1 promoter (P (AOX1) ) is still unclear. A Cys(2)His(2) zinc finger transcriptional repressor PpNrg1 localized to nucleus and participated in the repression of P (AOX1) in P. pastoris in glucose and glycerol. Quantitative real-time PCR revealed that PpNrg1 repressed expression of numerous genes involved in methanol utilization and peroxisome biogenesis in 0.02 % glucose and 1 % (v/v) glycerol. Electrophoretic mobility shift assay and DNase I footprinting assay revealed that PpNrg1 bound to five sites of P (AOX1) , including two binding sites of PpMxr1, which is an indispensable activator of P (AOX1) in P. pastoris. Transcriptional repressor PpNrg1 suppresses P (AOX1) in glucose and glycerol by directly binding to five sites of P (AOX1) , including two binding sites of transcriptional activator PpMxr1.