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Simple Summary: The β-defensin-1 (BD-1) is rich in disulfide bonds and antibacterial peptides with direct bactericidal function activity. Equine BD-1 was initially reported in 2004, however, there are no reports on the specific mechanisms that characterize the impact of Equine BD-1 on innate immune function. In this study, we map the tissue distribution of Equus BD-1 (i.e., Equine BD-1, ass BD-1, and mule BD-1) and compare their expression levels in various tissues, and we showed that Equine BD-1, ass BD-1, and mule BD-1 have an identical (100%) open reading frame (ORF). Next, we expressed the ORF of Equus BD-1 sequence fragment (OEBD-1) in the E. coli expression system and used this to treat an S. aureus-infected murine macrophage cell line in vitro. Results indicated that the OEBD-1 increased cytokines expression and associated signaling pathway, furthermore, OEBD-1 promoted macrophage phagocytose S. aureus in vitro. The report provides a theoretical basis for the development of defensins as potential alternatives to antibiotics. Beta-defensin-1 (BD-1) is among the class of antibacterial peptides that are rich in disulfide bonds, have direct antibacterial activity and showed enhanced expression following external stimulation. However, existing research studies only treated BD-1 to cell models without stimulation from pathogen-associated molecular patterns (PAMPs), which will further influence our understanding of the role of BD-1. In this study, we map the tissue distribution of Equus BD-1 (i.e., eBD-1, ass BD-1, and mule BD-1) and compare their expression levels in various tissues. We further characterize the three kinds of Equus BD-1 by analyzing their full-length cDNA. We showed that eBD-1, ass BD-1, and mule BD-1 have an identical (100%) open reading frame (ORF). The ORF encoding OEBD-1 expressed the ORF in the E. coli Top10 expression system. This expression system was combined with an S. aureus-infected J774A.1 macrophage cell line to determine the influence on innate immune mediator expression. Using this expression model system, it was determined that the OEBD-1 protein enhanced IL-6 and TNF-α secretion. It can also promote TLR2, IL-1β, CCL2, CCL7, CXCL10 and NF-κB p65 mRNA expression. Moreover, OEBD-1 upregulates phosphorylation of ATK, Syk and IκB-α. In addition, OEBD-1 enhances the macrophage's ability to phagocytose S. aureus. In conclusion, Equus BD-1 was shown to play an essential role in macrophage-involved innate immune responses in an in vitro system. [ABSTRACT FROM AUTHOR]