Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical regulator of adipogenesis. Our previous study showed that unlike human and mouse PPARγ transcripts, several chicken PPARγ transcript variants contain upstream open reading frames (uORFs) in their 5′ untranslated region (5′UTR). To decipher the role of uORFs in post-transcriptional regulation of chicken PPARγ gene, we constructed wild-type (psiCHECK2-cPPARγ3-5′UTR-WT) and a uORF mutant (the upstream ATG (uATG) was mutated to stop codon TGA) 5′UTR reporters (psiCHECK2-cPPARγ3- 5′UTR-Mut) of chicken PPARγ transcript variant 3 (cPPARγ3). These two reporters were individually transfected into immortalized chicken pre-adipocytes (ICPA) and DF1 cells, and the renilla luciferase (hRluc) activity and mRNA expression level were detected by reporter assay and qRT-PCR. The results showed that the hRluc activity of the mutated 5′UTR was significantly higher than that of the wild-type 5′UTR in ICPA cells (P<0.01), and the hRluc activity of the mutated 5′UTR tended to be higher than that of the wild-type 5′UTR in DF1 cells, but this difference did not reach statistical significance (P>0.05). The qRT-PCR analysis showed, in ICPA cells, the hRluc mRNA expression was significantly lower in the cells transfected with the mutated 5′UTR construct than in the cells transfected with the wild-type 5′UTR construct (P<0.01). In DF1 cells, the hRluc mRNA expression tended to be lower in the cells transfected with the mutated 5′UTR construct than in the cells transfected with the wild-type 5′UTR construct, but this difference did not reach statistical significance (P>0.05). To further gain insight into the post-transcriptional regulation of cPPARγ3 by the uORF, we constructed the expression plasmids bearing the full-length coding region of chicken PPARγ gene plus either wild-type or mutant uORF 5′UTR (pcDNA3.1-cPPARγ3-WT and pcDNA3.1-cPPARγ3-Mut). These two constructed PPARγ expression plasmids were individually transiently transfected into both ICPA and DF1 cells, and PPARγ mRNA and protein levels were assayed by qRT-PCR and western blotting. The result showed that in both cell lines, PPARγ mRNA expression was significantly lower in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.05). In contrast, western blot analysis showed that PPARγ protein level was significantly higher in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.001). Taken together, our results demonstrate that the uORF in 5′UTR of the cPPARγ3 inhibits its translation.