Mitochondrial inner membrane uncoupling proteins (UCP) dissipate the proton electrochemical gradient established by the respiratory chain, thus affecting the yield of ATP synthesis. UCP overexpression in plants has been correlated with oxidative stress tolerance, improved photosynthetic efficiency and increased mitochondrial biogenesis. This study reports the main transcriptomic responses associated with the overexpression of an UCP (AtUCP1) in tobacco seedlings. Compared to wild-type (WT), AtUCP1 transgenic seedlings showed unaltered ATP levels and higher accumulation of serine. By using RNA-sequencing, a total of 816 differentially expressed genes between the investigated overexpressor lines and the untransformed WT control were identified. Among them, 239 were up-regulated and 577 were down-regulated. As a general response to AtUCP1 overexpression, noticeable changes in the expression of genes involved in energy metabolism and redox homeostasis were detected. A substantial set of differentially expressed genes code for products targeted to the chloroplast and mainly involved in photosynthesis. The overall results demonstrate that the alterations in mitochondrial function provoked by AtUCP1 overexpression require important transcriptomic adjustments to maintain cell homeostasis. Moreover, the occurrence of an important cross-talk between chloroplast and mitochondria, which culminates in the transcriptional regulation of several genes involved in different pathways, was evidenced.