Lactoferrin is a multifunctional, iron-binding glycoprotein found in physiological fluids of mammals. Many functions of lactoferrin are thought to be involved in the N-lobe. In the present study, a gene encoding the N-lobe of porcine lactoferrin was cloned and expressed in Pichia methanolica. The gene contains 1038 bp, an “AGG” codon between 1039-1041bp in the PLF gene was replaced with a “TAA” stop codon through primer design and PCR. The sequencing results were aligned with the sequence (Genbank, L77887), indicating that the homology between these two sequences was 99%. The porcine lactoferrin N-lobe (PLfN) was expressed successfully in Pichia methanolica, and was secreted into the culture medium. The protein translated from the new ORF contained 346 amino acid residues, including a 17-amino acid signal peptide, its MW was approximately 42 kD, and it possessed antibacterial activity toward Escherichia coli ATCC25922. The method established in our study will pave the way for efficient industrial production of recombinant PLfN on a large scale for future utilization of this protein as a feed additive.