Objective <p>Beta-defensins, a group of antimicrobial peptides that can modulate immune response, are encoded by genes (DEFBs) on chromosome 8p23.1, a region subject to copy number variations (CNVs). This study aimed to investigate the influence of <i>DEFB4</i> CNV on both mRNA and protein expression in human monocytes.</p> Results <p>A cohort of 862 healthy blood donors was screened for <i>DEFB4</i> copy number (CN) by paralog ratio test and 66 individuals were recalled for a second blood donation. Monocytes were isolated from peripheral blood and stimulated by LPS. <i>DEFB4</i> mRNA and β-defensin 2 protein levels were quantified using qPCR and ELISA, respectively. <i>DEFB4</i> CN was precisely determined via ddPCR. <i>DEFB4</i> mRNA was undetectable in unstimulated monocytes but inducible by LPS. Beta-defensin 2 was detectable in freshly isolated monocytes. After stimulation with LPS, beta-defensin 2 was strongly induced and the level was significantly higher than that of non-stimulated controls. A significant correlation between <i>DEFB4</i> CN and mRNA expression level was found (<i>P</i> &lt; 0.05, r_s = 0.25). However, no correlation between <i>DEFB4</i> CN and protein expression including baseline level and the level after stimulation was found (<i>P</i> &gt; 0.05). In conclusion, <i>DEFB4</i> CNV has an impact on gene expression in mRNA but not protein level in monocytes.</p>