Peroxisome proliferator activated receptor gamma (PPARγ) is a master regulator in lipid metabolism and widely exists in vertebrates. However, the molecular structure and transcriptional activity of PPARγ in fish are still unclear. This study cloned PPARγ from Nile tilapia (Oreochromis niloticus) referred as NtPPARγ and transfected the NtPPARγ plasmids into HEK-293 cells to explore its mechanism of transcriptional regulation in fish. The expression of NtPPARγ was compared in fed and fasted fish. Two transcripts of NtPPARγ varied at the 5'-untranslated region and the DNA binding domain was highly conserved. Thirty-nine amino acid residues in the ligand binding domain in Nile tilapia were different from those in human. Two transcripts showed different expression profiles in 11 tissues, but both were highly expressed in liver, intestine and kidney. The transcriptional activity assay showed that NtPPARγ collaborates with retinoid X-receptor α (NtRXRα) to regulate the expression of Nile tilapia fatty acid binding protein 4 (FABP4), the compartment of which have been identified as the target gene of PPARγ in human. In the fish fasting trial, the mRNA expression of NtPPARγ1 and NtPPARγ2 in intestine and liver at 3. h post-feeding (HPF) was lower than those at 8 HPF, 24 HPF and in fish fasted for 36. h, but was relatively stable in kidney among different feeding treatments. In conclusion, the DNA binding domain in PPARγ was highly conserved, while the ligand binding domain was moderately conserved. In Nile tilapia, the PPARγ collaborates with RXRα to perform transcriptional regulation of FABP4 at least in vitro. The plasmid system established in this study along with a cell line from Nile tilapia will be useful tools for the further functional study of PPARγ in fish.