β-Defensins are cationic, antimicrobial peptides that participate in antimicrobial defense as well as the regulation of innate and adaptive immunity. Human β-defensin 126 (DEFB126) is a multifunctional glycoprotein consisting of a conserved β-defensin core and a unique long glycosylated peptide tail. The long glycosylated peptide tail has been proven to be critical for efficient transport of sperm in the female reproductive tract, preventing their immune recognition, and efficient delivery of capacitated sperm to the site of fertilization. However, the functions of the conserved β-defensin core remain to be fully elucidated. In the present work, the conserved β-defensin core of the DEFB126 was expressed to explore its potential antimicrobial and anti-inflammatory activities. The DEFB126 core peptide exhibited both high potency for binding and neutralizing lipopolysaccharide (LPS) in vitro, and potent anti-inflammatory ability by down-regulating the mRNA expression of pro-inflammatory cytokines including IL-α, IL-1β, IL-6 and TNF-α in a murine macrophage cell line RAW264.7. The treatment with the DEFB126 core peptide also led to correspondingly decreased secretion of IL-6 and TNF-α. The blockade of LPS-induced p42/44 and p38 MAPK signal pathway might contribute to the anti-inflammation effects of the DEFB126 core peptide. Furthermore, fluorescence-labeled DEFB126 could enter RAW 264.7 cells and reduce the production of LPS-stimulated inflammatory factors, implying that DEFB126 might also participate in intracellular regulation beyond its direct LPS neutralization. In summary, our results demonstrate that the DEFB 126 core peptide has critical functions in parallel to its C-terminal tail by showing LPS-binding activity, anti-inflammatory effects and intracellular regulatory function.