A BoIFN-α gene that included signal sequence was amplified from bovine liver genomic DNA. The gene was named BoIFN-α 14 according to the position at which the encoded gene of the bovine IFN was located in the bovine genome. The sequence included a 23-amino acid signal peptide and 166-amino acid mature peptide. The structural characteristics and phylogenetic relationships of the BoIFN-α 14 gene were analyzed. A recombinant mature BoIFN-α 14 (rBoIFN-α 14 ) was expressed in the yeast Pichia pastoris. Antiviral activity, antiproliferation activity and physicochemical characteristics were determined in vitro. Recombinant BoIFN-α 14 exhibited a considerable antiviral activity against Vesicular stomatitis virus (VSV), which was neutralized by a rabbit polyclonal anti-rBoIFN-α antibody, and could inhibit cell proliferation. It was also found to be highly sensitive to trypsin and stable at pH 2.0 or 65°C. This study revealed that rBoIFN-α 14 has the typical characteristics of IFN-α and can be used for both research and industrial application.