Bovine interferon-omega3 (BoIFN-ω3) gene was amplified from bovine liver genomic DNA， which encodes a 195-amino acid protein containing a 23-amino acid signal peptide. Analysis of the molecular characteristics revealed that BoIFN-ω3 evolving from IFN-ω， contained four cysteine residues and five alpha helices， showing that BoIFN-ω3 presented the typical molecular characteristics of type I interferon. BoIFN-ω3 exhibited antiviral and antiproliferative activities， which exerted a protective effect against VSV in several mammalian cell lines， as well as against BEV， IBRV， and BVDV in MDBK cell. Moreover， BoIFN-ω3 was shown to be highly sensitive to trypsin， but remaining stable despite changes in pH and temperature. Additionally， BoIFN-ω3 induced the transcription of Mx1， ISG15， and ISG56 genes， as well as the expression of Mx1 protein in a time-dependent manner. These findings will be useful to further study BoIFN-ω in host's defence against infectious diseases， particularly viral infections. Furthermore， results will facilitate further research on the bovine interferon family.