In this report, we studied DHX32 expression in human Jurkat T cells. Co-stimulation of CD3 and CD28 resulted in upregulation of DHX32. No significant changes in the expression of the closely related RNA helicases were seen. Ionomycin treatment alone was sufficient to upregulate the expression of DHX32 mRNA isoform transcribed from the proximal promoter. We cloned DHX32 proximal promoter and identified a 218bp fragment containing two potential binding sites for the transcription factor nuclear factor of activated T cells (NF-AT). Mutation of core sequence of NF-AT resulted in reduced transcriptional activity, with more reduction observed in the second NF-AT site. Electrophoretic mobility shift assay results were consistent with a specific binding of NF-AT from ionomycin stimulated nuclear extract of Jurkat cells to oligonucleotide probes from DHX32 proximal promoter. These results suggest that the DHX32 expression is modulated in Jurkat T cells via a pathway that involves NF-AT.