Transcription factor GATA4 is expressed in somatic cells of the mammalian testis. Gene targeting studies in mice have shown that GATA4 is essential for proper differentiation and function of Sertoli cells. The role of GATA4 in Leydig cell development, however, remains controversial, because targeted mutagenesis experiments in mice have not shown a consistent phenotype, possibly due to context-dependent effects or compensatory responses. We therefore undertook a reductionist approach to study the function of GATA4 in Leydig cells. Using microarray analysis and quantitative RT-PCR, we identified a set of genes that are downregulated or up-regulated after small interfering RNA (siRNA)-mediated silencing of Gata4 in the murine Leydig tumor cell line mLTC-1. These same genes were dysregulated when primary cultures of Gata4flox/flox adult Leydig cells were subjected to adenovirus-mediated cre-lox recombination in vitro. Among the down-regulated genes were enzymes of the androgen biosynthetic pathway (Cyp11a1, Hsd3b1, Cyp17a1, and Srd5a). Silencing of Gata4 expression in mLTC-1 cells was accompanied by reduced production of sex steroid precursors, as documented by mass spectrometric analysis. Comprehensive metabolomic analysis of GATA4-deficient mLTC-1 cells showed alteration of other metabolic pathways, notably glycolysis. GATA4-depleted mLTC-1 cells had reduced expression of glycolytic genes (Hk1, Gpi1, Pfkp, and Pgam1), lower intracellular levels of ATP, and increased extracellular levels of glucose. Our findings suggest that GATA4 plays a pivotal role in Leydig cell function and provide novel insights into metabolic regulation in this cell type.