The NF-κB signaling pathway, which plays an important role in cell fate determination in various cells, has been found to be involved in the activation of long clusters of enhancers known as super-enhancers (SEs) for transcriptional regulation. However, the contribution of NF-κB to SEs has not yet been validated under microscopic observation. Using fluorescence imaging, single-cell transcriptome, and chromatin accessibility analyses, we show that NF-κB subunit RelA nuclear foci formation and single-cell gene expression demonstrate SE-like properties in anti-IgM-stimulated B cells. This contributed to bimodal and enhanced cell-to-cell variability in transcriptional response. Furthermore, we found that the predicted cis-regulatory interacting genomic regions from chromatin co-accessibility analysis were associated with the observed transcriptional heterogeneity. These findings suggest that NF-κB-mediated SE formation is important for the expression of NF-κB target genes and the amplification of transcriptional heterogeneity in response to environmental stimuli in B cells.