Recent data indicate that the deficiencies of several pro-inflammatory cytokine genes affect skin wound healing processes. Hence, we explored skin wound healing processes in mice deficient in IL-1 receptor antagonist (IL-1ra), a potent endogenous antagonist against a pro-inflammatory cytokine, IL-1. Wound closure was delayed with attenuated collagen accumulation in IL-1ra knockout (KO) mice, compared with wild-type (WT) mice. On the contrary, leukocyte recruitment was exaggerated with augmented IL-1 expression in IL-1ra KO mice, implying that IL-1ra deficiency enhanced local inflammatory reaction at the wound sites. Consistently, nuclear translocation of a pro-inflammatory transcription factor, NF-κB, was significantly enhanced and prolonged in fibroblasts in IL-1ra KO mice, compared with WT mice. Previous in vitro observations demonstrated that NF-κB activation could attenuate transforming growth factor (TGF)-β/Smad signaling pathway, which has crucial roles for collagen deposition in wound healing processes. Indeed, in IL-1ra KO mice, the TGF-β/Smad signaling pathway was suppressed as evidenced by decreases in phosphorylated Smad2/3 and a reciprocal increase in Smad7 at the wound sites, compared with WT mice. These results demonstrated that the absence of IL-1ra resulted in aberrant NF-κB activation and reciprocal diminution in TGF-β/Smad signaling and eventually attenuated collagen deposition during skin wound healing in vivo.