Adipose tissue is currently being recognized as an important endocrine organ, carrying defects in a number of metabolic diseases. Mitochondria play a key role in normal adipose tissue function and mitochondrial alterations can result in pathology, like lipodystrophy or type 2 diabetes. Although Pgc1α is regarded as the main regulator of mitochondrial function, downstream Nrf1 is the key regulator of mitochondrial biogenesis. Nrf1 is also involved in a wide range of other processes, including proliferation, innate immune response, and apoptosis. To determine transcriptional targets of Nrf1, 3T3-L1 preadipocytes were transfected with either pNrf1 or a control vector. Two days post-confluence, 3T3-L1 preadipocytes were allowed to differentiate. At day 8 of differentiation, Nrf1 overexpressing cells had an increased mtDNA copy number and reduced lipid content. This was not associated with an increased ATP production rate per cell. Using global gene expression analysis, we observed that Nrf1 overexpression stimulated cell proliferation, apoptosis, and cytokine expression. In addition, prolonged Nrf1 induced an adipokine expression profile of insulin resistant adipocytes. Nrf1 has a wide range of transcriptional targets, stimulators as well as inhibitors of adipose tissue functioning. Therefore, post-transcriptional regulation of Nrf1, or stimulating specific Nrf1 targets may be a more suitable approach for stimulating mitochondrial biogenesis and treating adipose tissue defects, instead of directly stimulating Nrf1 expression. In addition, our results show that short-term effects can drastically differ from long-term effects.