Immunosuppressive neutrophils (PMN‐II) appearing in association with burn injury have a role on the increased susceptibility of burn patients to various infections. In the present study, the role of PMN‐II on the production of human β‐defensins (HBDs), important molecules on host antimicrobial innate immunities, by human keratinocytes was studied. Normal human epidermal keratinocytes (NHEKs) were cultured with neutrophils (PMNs) isolated from burn patients or healthy volunteers in dual‐chamber transwells. Culture fluids harvested 24 h after cultivation were assayed for HBDs using enzyme‐linked immunosorbent assay. Also, these culture fluids were assayed for their antimicrobial activities by a standard colony‐counting method using Pseudomonas aeruginosa. In the results, PMNs isolated from peripheral blood of burn patients were confirmed as PMN‐II, because these cells produced CC‐chemokine ligand 2 (CCL2), but not interleukin (IL)‐12 and CC‐chemokine ligand 3 (CCL3). Culture fluids of NHEK transwell‐cultured with healthy PMNs exhibited strong killing activities against P. aeruginosa (96% inhibition), however, the growth of bacteria was not dramatically inhibited by the culture fluids of NHEK transwell‐cultured with burn‐patient PMNs (36% inhibition). IL‐12 and CCL3 containing culture fluids of healthy PMNs stimulated with the bacterial antigen or rCCL3 and rIL‐12 enhanced the production of HBD2 and HBD3 by NHEK, whereas CCL2 containing culture fluids of burn‐patient PMN stimulated with the antigen or rCCL2 inhibited the HBD production by NHEK. These results indicate that PMN‐II appearing in association with burn injury contribute to the decreased production of HBDs in thermally injured patients.