Human alpha-defensin 5 (HD5), a small cysteine-rich peptide expressed predominantly in small intestine and female reproductive tissues, plays an important role in innate and adaptive immunity. It is a worthy yet challenging work to produce bioactive recombinant HD5 through the use of bioengineering techniques. Here, we present the expression and purification of recombinant HD5 mature peptide (rmHD(5)) in Pichia pastoris. To avoid generating unfavorable extra N-terminal amino acids, Red/ET homologous recombination was applied to construct the expression vector pPIC9K-mHD(5) by insertion of a polymerase chain reaction-amplified DNA fragment coding for mHD(5) into the plasmid pPIC9K, at a position right after the cleavage sequence of Kex2. The pPIC9K-mHD(5) vector was transformed into P. pastoris GS115 cells, and positive colonies harboring genomic integration of the multicopy mHD(5) nucleotide sequence were screened out and used for fermentation. After high-cell density fermentation of P. pastoris GS115-HD5, a two-step purification strategy of macroporous resin adsorption chromatography followed by cation exchange chromatography was performed to obtain purified rmHD(5). The results showed that about 165.0 mg/l of rmHD(5) with its intact N-terminal amino acid sequence as revealed by mass spectrometry analysis and amino acid sequencing was produced under optimal bioreactor-culture conditions and that approximately 50% of the initial rmHD(5) was recovered after purification. The in vitro experiments revealed that rmHD(5) exhibited a prominent antibacterial activity and potency to block human papillomavirus infection. This is the first report on the production and purification of bioactive rmHD(5) in P. pastoris. This study also provides considerations for production of other antimicrobial peptides using the P. pastoris expression system.