NF-kappaB signaling is known to be critically regulated by the NF-kappaB-inducible inhibitor protein IkappaBalpha. The resulting negative feedback has been shown to produce a propensity for oscillations in NF-kappaB activity. We report integrated experimental and computational studies that demonstrate that another IkappaB isoform, IkappaBepsilon, also provides negative feedback on NF-kappaB activity, but with distinct functional consequences. Upon stimulation, NF-kappaB-induced transcription of IkappaBepsilon is delayed, relative to that of IkappaBalpha, rendering the two negative feedback loops to be in antiphase. As a result, IkappaBepsilon has a role in dampening IkappaBalpha-mediated oscillations during long-lasting NF-kappaB activity. Furthermore, we demonstrate the requirement of both of these distinct negative feedback regulators for the termination of NF-kappaB activity and NF-kappaB-mediated gene expression in response to transient stimulation. Our findings extend the capabilities of a computational model of IkappaB-NF-kappaB signaling and reveal a novel regulatory module of two antiphase negative feedback loops that allows for the fine-tuning of the dynamics of a mammalian signaling pathway.