Noise in gene expression is a main determinant of phenotypic variability. Increasing experimental evidence suggests that genome-wide cellular constraints largely contribute to the heterogeneity observed in gene products. It is still unclear, however, which global factors affect gene expression noise and to what extent. Since eukaryotic gene expression is an energy demanding process, differences in the energy budget of each cell could determine gene expression differences. Here, we quantify the contribution of mitochondrial variability (a natural source of ATP variation) to global variability in gene expression. We find that changes in mitochondrial content can account for ∼50% of the variability observed in protein levels. This is the combined result of the effect of mitochondria dosage on transcription and translation apparatus content and activities. Moreover, we find that mitochondrial levels have a large impact on alternative splicing, thus modulating both the abundance and type of mRNAs. A simple mathematical model in which mitochondrial content simultaneously affects transcription rate and splicing site choice can explain the alternative splicing data. The results of this study show that mitochondrial content (and/or probably function) influences mRNA abundance, translation, and alternative splicing, which ultimately affects cellular phenotype.[Supplemental material is available for this article.] Cellular heterogeneity can result from noise generated during gene expression and plays an essential role in fundamental processes such as development, cell differentiation, and cancer (Raj and van Oudenaarden 2008;Eldar and Elowitz 2010;Balázsi et al. 2011). Gene expression noise may originate from stochasticity in the biochemical reactions at an individual gene (intrinsic noise) or from fluctuations in cellular components inducing a global effect (extrinsic noise) (Elowitz et al. 2002;Maheshri and O'Shea 2007). Extrinsic noise is often a dominant source of variation both in prokaryotes (Taniguchi et al. 2010) and eukaryotes (Raser and O'Shea 2004;Newman et al. 2006). Despite this, the origins of extrinsic fluctuations are mostly unknown, although random protein partitioning from cell growth and division (Rosenfeld et al. 2005;Volfson et al. 2006), upstream transcription factors (Volfson et al. 2006), or cell cycle stage (Zopf et al. 2013) have been shown to contribute to variability in protein levels. A common constraint across eukaryotic gene expression is its high energy cost (with ∼75% of the ATP cellular energy budget invested into mRNA and protein polymerization) (Forster et al. 2003;Wagner 2005;Lane and Martin 2010), where every step, from chromatin remodeling to transcription elongation, assembly of splicing factors, and translation, depends on energy (Fig. 1A). Since most of the energy required in normal cells is supplied by mitochondrial oxidative phosphorylation (Vander Heiden et al. 2009), variability in the number and/or functionality of mitochondria is a natural source of variability in ATP content...