The Wilms' tumor gene, WT1, encodes a zinc finger transcription factor which functions both as a tumor suppressor and a key regulator of embryonic development. WT1 is also expressed in hematopoietic progenitor cells and has been implicated in the pathogenesis of acute myeloid leukemia. The objective of this study was to explore a previously unrecognized role of WT1 in immune function and to identify candidate target genes involved herein. WT1 mRNA could be detected by real-time reverse transcription PCR in monocytes from healthy volunteers and in several macrophagocytic cell lines. Silencing of WT1 by siRNA transfection reduced interleukin (IL)-10 mRNA to approximately 20% of the level obtained upon transfection with a non-targeting siRNA. Correspondingly, IL-10 transcripts were increased >15-fold by forced expression of WT1 in different cell lines. In contrast, transfection of an alternatively spliced WT1 protein, which has a role in mRNA processing rather than in transcriptional regulation, did not significantly change IL-10 mRNA levels. A reporter construct containing a luciferase gene under control of the murine IL-10 promoter was stimulated more than 5-fold by transient co-transfection of RAW 264.7 macrophages with a WT1 expression plasmid. The responsible WT1 binding site was located to the proximal IL-10 promoter by the use of several 5' truncated and mutated constructs. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) confirmed the binding of WT1 protein to the identified regulatory element. These findings suggest a novel role of WT1 in the innate immune response. Transcriptional activation of IL-10, which acts mainly as an anti-inflammatory cytokine, by WT1 might be important for the escape of WT1-expressing tumor cells from immune surveillance.