Ice-binding proteins (IBPs) can bind to the ice crystal and inhibit its growth. Because this property of IBPs can increase the freeze-thaw survival of cells, IBPs have attracted the attention from industries for their potential use in biotechnological applications. However, their use was largely hampered by the lack of the large-scale recombinant production system. In this study, the codon-optimized IBP from Leucosporidium sp. (LeIBP) was constructed and subjected to high-level expression in methylotrophic Pichia pastoris system. In a laboratory-scale fermentation (7 L), the optimal induction temperature and pH were determined to be 25 A degrees C and 6.0, respectively. Further, employing glycerol fed-batch phase prior to methanol induction phase enhanced the production of recombinant LelBP (rLeIBP) by similar to 100 mg/l. The total amount of secreted proteins at these conditions (25 A degrees C, pH 6.0, and glycerol fed-batch phase) was similar to 443 mg/l, 60 % of which was rLeIBP, yielding similar to 272 mg/l. In the pilot-scale fermentation (700 L) under the same conditions, the yield of rLeIBP was 300 mg/l. To our best knowledge, this result reports the highest production yield of the recombinant IBP. More importantly, the rLeIBP secreted into culture media was stable and active for 6 days of fermentation. The thermal hysteresis (TH) activity of rLeIBP was about 0.42 A degrees C, which is almost the same to those reported previously. The availability of large quantities of rLeIBP may accelerate further application studies.