Active brown adipose tissue is responsible for non-shivering thermogenesis in mammals which affects energy homeostasis. The molecular mechanisms underlying this activation as well as the formation and activation of brite adipocytes have gained increasing interest in recent years as they might be utilized to regulate systemic metabolism. We show here that the transcriptional regulators SRF and MKL1 both act as repressors of brown adipogenesis. Loss-of-function of these transcription factors leads to a significant induction of brown adipocyte differentiation, increased levels of UCP1 and other thermogenic genes as well as increased respiratory function, while SRF induction exerts the opposite effects. Interestingly, we observed that knockdown of MKL1 does not lead to a reduced expression of typical SRF target genes and that the SRF/MKL1 inhibitor CCG-1423 had no significant effects on brown adipocyte differentiation. Contrary, knockdown of MKL1 induces a significant increase in the transcriptional activity of PPARγ target genes and MKL1 interacts with PPARγ, suggesting that SRF and MKL1 independently inhibit brown adipogenesis and that MKL1 exerts its effect mainly by modulating PPARγ activity.