Insulin-like 3 (INSL3) is a hormone produced by testicular Leydig cells throughout life. During embryonic life it regulates an essential step of testicular descent, whereas in adults it acts as a male germ cell survival factor. Despite the importance of INSL3 for male sex differentiation and function, very little is known regarding the molecular mechanisms that regulate its expression. So far, the nuclear receptor SF-1 is the only transcription factor known to regulate the mouse Insl3 promoter in Leydig cells. In order to further our understanding of the transcriptional regulation of INSL3 expression, we have isolated the human INSL3 promoter and tested the effects of the nuclear receptors SF-1, LRH-1, and Nur77 on its activity in Leydig cells. In transfections assays, all three nuclear receptors activated the human INSL3 promoter but especially Nur77, which acted through a novel regulatory element. Thus, the human INSL3 promoter constitutes a novel target for the orphan nuclear receptor Nur77.