In this study, we performed high throughput RNA sequencing at the preadipocyte (D0) and differentiated adipocyte (D7) stages of primary brown preadipocyte differentiation in order to characterize the transcriptional events regulating differentiation and function. Compared to the preadipocyte stage (D0), 6,668 genes were identified as differentially expressed genes (DEGs) with a fold change of ≥ 2.0 at the differentiated adipocyte stage (D7). Several adipogenic genes including peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer-binding protein-α (C/EBPα), and Krüppel-like factor (KLF) family genes were differentially expressed at D0 and D7. Since KLF16 gene expression was downregulated at day 7 and its adipogenic function has not been characterized, we investigated its role in adipogenesis. Knockdown of KLF16 stimulated the differentiation of both brown and 3T3-L1 preadipocytes, and led to increased PPARγ expression. However, overexpression of KLF16 had opposite effects. Furthermore, KLF16 downregulated PPARγ expression in brown adipocytes and inhibited its promoter activity. These results indicate that KLF16 inhibits adipogenesis through downregulation of PPARγ expression.