Hydrogen peroxide (H₂O₂) has been implicated in the regulation of the transcription factor NF-κB, a key regulator of the inflammatory process and adaptive immunity. However, no consensus exists regarding the regulatory role played by H₂O₂. We discuss how the experimental methodologies used to expose cells to H₂O₂ produce inconsistent results that are difficult to compare, and how the steady-state titration with H₂O₂ emerges as an adequate tool to overcome these problems. The redox targets of H₂O₂ in the NF-κB pathway—from the membrane to the post-translational modifications in both NF-κB and histones in the nucleus—are described. We also review how H₂O₂ acts as a specific regulator at the level of the single gene, and briefly discuss the implications of this regulation for human health in the context of κB polymorphisms. In conclusion, after near 30 years of research, H₂O₂ emerges not as an inducer of NF-κB, but as an agent able to modulate the activation of the NF-κB pathway by other agents. This modulation is generic at the level of the whole pathway but specific at the level of the single gene. Therefore, H₂O₂ is a fine-tuning regulator of NF-κB-dependent processes, as exemplified by its dual regulation of inflammation. Antioxid. Redox Signal. 11, 2223–2243.