Objectives To identify the zinc transport function of the membrane proteins Gt1 and Zrt1 inKomagataella phaffii(Pichia pastoris) and study their regulatory mode. Results Two membrane proteins that might have zinc transport function were found inK. phaffii.GT1was known to encode a glycerol transporter belonging to the Major Facilitator Superfamily.ZRT1was predicted to resemble the zinc transporter gene inSaccharomyces cerevisiae. Consistent with the prediction, protein plasma-membrane localizations were confirmed by ultracentrifugation and confocal microscopy. Their zinc binding abilities were identified by ITC in vitro, and the impaired zinc uptake activity caused by their deficiencies was confirmed by zinc fluorescence quantification in vivo. Furthermore, zinc excess could turn the two channels off, while zinc deficiency induced their expressions. Gt1 could only function to maintain zinc homeostasis in glycerol, while the block of Gt1 function might lead to Zrt1 upregulation in glucose. Conclusions The zinc transport capabilities of Gt1 and Zrt1 were identified in vivo and in vitro. Their regulatory mode to maintain zinc homeostasis inK. phaffiiis a new inspiration.