Description: Knockdown of the transcription factors GATA4 and GATA6 in granulosa cells (GCs) impairs folliculogenesis and induces infertility. To investigate the pathways and genes regulated by these factors, we performed microarray analyses on wild-type GCs or GCs lacking GATA4, GATA6, or GATA4/6 (G4 gcko , G6 gcko , and G4/6 gcko ) after in vivo treatment with equine chorionic gonadotropin. GATA4 deletion affected a greater number of genes than GATA6, which correlates with the subfertility observed in G4 gcko mice and the normal reproductive function found in G6 gcko animals. An even greater number of genes were affected by the deletion of both factors. Moreover, the expression of FSH receptor, LH receptor, inhibin α and β, versican, pregnancy-associated plasma protein A, and the regulatory unit 2b of protein kinase A, which are known to be crucial for ovarian function, was greatly affected in double GATA4 and GATA6 knockouts when compared with single GATA-deficient animals. This suggests that GATA4 and GATA6 functionally compensate for each other in the regulation of key ovarian genes. Functional enrichment revealed that ovulation, growth, intracellular signaling, extracellular structure organization, gonadotropin and growth factor actions, and steroidogenesis were significantly regulated in G4/6 gcko mice. The results of this analysis were confirmed using quantitative polymerase chain reaction, immunohistochemical, and biological assays. Treatment of GCs with cAMP/IGF-I, to bypass FSH and IGF-I signaling defects, revealed that most of the affected genes are direct targets of GATA4/6. The diversity of pathways affected by the knockdown of GATA underscores the important role of these factors in the regulation of GC function.