Human β‐defensin 3 (hBD‐3) activates antigen‐presenting cells through Toll‐like receptors (TLRs) 1/2. Several TLR1/2 agonists have been identified but little is known about how they might differentially affect cellular activation. We compared the effects of hBD‐3 with those of another TLR1/2 agonist, Pam3CSK4, in human monocytes. Monocytes incubated with hBD‐3 or Pam3CSK4 produced interleukin‐6 (IL‐6), IL‐8 and IL‐1β, but only Pam3CSK4 induced IL‐10. The IL‐10 induction by Pam3CSK4 caused down‐modulation of the co‐stimulatory molecule, CD86, whereas CD86 expression was increased in monocytes exposed to hBD‐3. Assessment of signalling pathways linked to IL‐10 induction indicated that mitogen‐activated protein kinases were activated similarly by hBD‐3 or Pam3CSK4, whereas the non‐canonical nuclear factor‐κB pathway was only induced by Pam3CSK4. Our data suggest that the lack of non‐canonical nuclear factor‐κB signalling by hBD‐3 could contribute to the failure of this TLR agonist to induce production of the anti‐inflammatory cytokine, IL‐10, in human monocytes.