The control of the mitochondrial biogenesis in brown adipose tissue (BAT), as part of the thermogenesis program, is a complex process that requires the integration of multiple transcription factors to orchestrate mitochondrial and nuclear gene expression. Despite the knowledge of the role of sex hormones on BAT physiology, little is known about the effect of these hormones on the mitochondrial biogenic program. The aim of this study was to determine the effect of testosterone, 17β-estradiol and progesterone on the expression of nuclear factors involved in the control of mitochondrial biogenesis and thermogenic function such as pparγ, pgc1α, nrf1, gabpa and tfam, and also an inhibitor of PI3K-Akt pathway, recently found to be involved in the control of mitochondrial recruitment (pten). For this purpose, an in vitro assay using cell cultured brown adipocytes was used to address the role of steroid hormones, progesterone, testosterone and 17β-estradiol on the mRNA expression of these factors by real time PCR. Thus, 17β-estradiol seemed to exert a dual effect, activating the PI3K-Akt pathway by inhibiting pten mRNA expression, and also inhibiting nrf1 and tfam mRNA expression. Progesterone seemed to positively stimulate mitochondriogenesis and BAT differentiation by increasing the mRNA expression of the gabpa-tfam axis and pparγ respectively, but also exerted a negative output by increasing pten mRNA levels. Finally, testosterone inhibited the transcription of pgc1α, the master factor involved in UCP1 expression and mitochondrial biogenesis. In conclusion, our results support the idea that sex hormones have direct effects on different mediators of the mitochondriogenesis program.