Intense resistance exercise causes a significant inflammatory response. NF-κB has been identified as a prospective key transcription factor mediating the postexercise inflammatory response. The purpose of this study was to determine whether a single bout of intense resistance exercise regulates NF-κB signaling in human skeletal muscle. Muscle biopsy samples were obtained from the vastus lateralis of five recreationally active, but not strength-trained, males (21.9 ± 1.3 yr) prior to, and at 2 and 4 h following, a single bout of intense resistance exercise. A further five subjects (4 males, 1 female) (23 ± 0.89 yr) were recruited as a nonexercise control group to examine the effect of the muscle biopsy protocol on key markers of skeletal muscle inflammation. Protein levels of IκBα and phosphorylated NF-κB (p65), as well as the mRNA expression of inflammatory myokines monocyte chemoattractant protein 1 (MCP-1), IL-6, and IL-8 were measured. Additionally, NF-κB (p65) DNA binding to the promoter regions of MCP-1, IL-6, and IL-8 was investigated. IκBα protein levels decreased, while p-NF-κB (p65) protein levels increased 2 h postexercise and returned to near-baseline levels by 4-h postexercise. Immunohistochemical data verified these findings, illustrating an increase in p-NF-κB (p65) protein levels, and nuclear localization at 2 h postexercise. Furthermore, NF-κB DNA binding to MCP-1, IL-6, and IL-8 promoter regions increased significantly 2 h postexercise as did mRNA levels of these myokines. No significant change was observed in the nonexercise control group. These novel data provide evidence that intense resistance exercise transiently activates NF-κB signaling in human skeletal muscle during the first few hours postexercise. These findings implicate NF-κB in the transcriptional control of myokines known to be central to the postexercise inflammatory response.