It is known that a number of transcription factors are key regulators in the complex process of adipocyte differentiation including peroxisome proliferator activated receptor γ (PPARγ) and the CCAAT enhancer binding protein α (C/EBPα). Studies have demonstrated that in pre-adipocyte 3T3-L1 cells constitutive expression of the DNA binding proteins GATA-2 and GATA-3 results in protein/protein interactions with C/EBPα resulting in down regulation of PPARγ and subsequent suppressed adipocyte differentiation with cells trapped at the pre-adipocyte stage. Thus it appears that GATA-2 and GATA-3 are of critical importance in regulating adipocyte differentiation through molecular interactions with PPARγ and C/EBPα. Recent reports suggest that berberine, an isoquinoline derivative alkaloid isolated from many medicinal herbs prevents differentiation of 3T3-L1 cells via a down regulation of PPARγ and C/EBPα expression. The aim of this study was to determine the effect of berberine on GATA-2 and 3 gene and protein expression levels during differentiation of 3T3-L1 cells. MTT (Methylthiazolyldiphenyl-tetrazolium bromide) was used to detect the cytotoxic effects of berberine on the viability of 3T3-L1 cells during proliferation and differentiation. Differentiation of 3T3-L1 cells was monitored by Oil Red O staining and RT-PCR of PPARγ and C/EBPα and the expression of GATA-2 and 3 was determined by RT-PCR and Western Blot. Results show that following treatment with 8μM berberine the mRNA and protein expression levels of GATA-2 and 3 were elevated and accompanied by inhibited adipocyte differentiation. These results may lead to the use of berberine to target the induction of specific genes such as GATA-2 and GATA-3 which affect adipocyte differentiation.