GATA4, a member of the GATA family, is a well-known transcription factor implicated in the regulation of sex determination and sexual differentiation in mammals. However, little is known about the possible role of GATA4 in fish reproduction. In the present study, a full-length GATA4 cDNA from the tilapia was cloned and characterized. The tilapia GATA4 gene contained an open reading frame (ORF) of 1179 nucleotides encoding a protein of 392 amino acids. Sequence alignment revealed that the tilapia GATA4 protein shared higher homology (ranging from 63.1 to 74.6%) with other vertebrates. RT-PCR analysis indicated that the GATA4 gene is expressed in the ovary, testis, liver, intestine and heart in adult tilapia. In situ hybridization was performed to examine the temporal and spatial expression patterns of GATA4 during tilapia gonadal differentiation and development. In the undifferentiated gonad, GATA4 was expressed in the somatic cells of both sexes. Subsequently, GATA4 expression persisted in the differentiated, juvenile and adult ovary and testis in tilapia. Our data indicate for the first time that GATA4 is not only necessary for the onset of gonadal differentiation, but also important for gonadal development and maturation.