Abstract The positive regulation of the NF-κB-signaling pathway in response to TCR stimulation has been well-studied. However, little is known about the negative regulation of this pathway in T cells. This negative regulation is crucial in controlling the duration of TCR signaling and preventing abnormal lymphocyte activation and proliferation. Therefore, understanding the negative regulation of TCR-mediated NF-κB signaling is essential in understanding the mechanisms involved in T cell function and homeostasis. TCR stimulation of human CD4+ T cells resulted in an increase in NF-κB2/p100 expression with no appreciable increase in p52, its cleavage product. Due to the presence of inhibitory ankyrin repeats in the unprocessed p100, this observation suggests that p100 may function as a negative regulator of the NF-κB pathway. Consistent with this hypothesis, ectopic expression of p100 inhibited TCR-mediated NF-κB activity and IL-2 production in Jurkat T cells. Conversely, knockdown of p100 expression enhanced NF-κB transcriptional activity and IL-2 production upon TCR activation. p100 inhibited the pathway by binding and sequestering Rel transcription factors in the cytoplasm without affecting the activity of the upstream IκB kinase. The kinetics and IκB kinase γ/NF-κB essential modulator dependency of p100 induction suggest that NF-κB2/p100 acts as a late-acting negative-feedback signaling molecule in the TCR-mediated NF-κB pathway.