Expression of the cytokine interleukin-13 (IL13) is critical for Th2 immune responses and Th2-mediated allergic diseases. Activation of human IL13 expression involves chromatin remodeling and formation of multiple DNase I-hypersensitive sites throughout the locus. Among these, HS4 is detected in the distal IL13 promoter in both naive and polarized CD4+ T cells. We show herein that HS4 acts as a position-independent, orientation-dependent positive regulator of IL13 proximal promoter activity in transiently transfected, activated human CD4+ Jurkat T cells and primary murine Th2 cells. The 3′-half of HS4 (HS4-3′) was responsible for IL13 up-regulation and bound nuclear factor (NF) 90 and NF45, as demonstrated by DNA affinity chromatography coupled with tandem mass spectrometry, chromatin immunoprecipitation, and gel shift analysis. Notably, the CTGTT NF45/NF90-binding motif within HS4-3′ was critical for HS4-dependent upregulation of IL13 expression. Moreover, transfection of HS4-IL13 reporter vectors into primary, in vitro differentiated Th2 cells from wild-type, NF45+/-, or NF90+/- mice showed that HS4 activity was exquisitely dependent on the levels of endogenous NF45 (and to a lesser degree NF90), because HS4-dependent IL13 expression was virtually abrogated in NF45+/- cells and reduced in NF90+/- cells. Collectively, our results identify NF45 and NF90 as novel regulators of HS4-dependent human IL13 transcription in response to T cell activation.