Since Jacob and Monod’s groundbreaking work on gene regulation, the lac operon has become a canonical example of how prokaryotic cells regulate the expression of genes in response to changes in environmental conditions [1]. The lac operon is responsible for the efficient metabolism of lactose in Escherichia coli bacteria ensuring that enzymes capable of digesting lactose are produced only when needed. The lac repressor-mediated DNA loop, which is formed when tetrameric lac repressor protein binds to two lac operator sites si- multaneously, is an important part of this gene regulatory network and is crucial for the repression of lac genes[2]. Long range genetic regulation by DNA looping, however, is not unique to the lac operon, but appears in a variety of contexts within prokaryotes, such as the ara or gal operons and is ubiquitous within eukaryotes [3]. While the biochemistry of these processes is generally well understood, the mechanics of the assembly and breakdown of protein-mediated DNA loops has only recently garnered much attention [4]. In this paper, we investigate the role that tension in the substrate DNA plays in the formation and break- down of protein-mediated DNA loops, and conclude that loop formation is acutely sensitive to entropic forces on the hundred-femtonewton scale. Protein-mediated DNA loop formation is driven by thermal fluctuations in the DNA which bring distant operators close enough for loop closure by a protein. However, it is quite surprising that the magnitude of these fluctuations, which one can estimate as k$_{B}$T/l$_{p}$ ≈ 80 fN where l$_{p}$ = 50 nm is the persistence length, is much smaller than the typical piconewton forces that arise in the intracellular environment, from molecular motors or DNA-cytoskeletal attachments for example. This observation has led to predictions that forces as small as a few hundred femtonewtons are sufficient to reduce the loop formation rate by more than two orders of magnitude [5–7]. Given that the cellular environment is thought to regularly subject DNA-protein complexes to large static or fluctuating forces, the cell must either use mechanical pathways to regulate genetic function, or compensate for the effects of tension to ensure the stable control of gene expression. To experimentally study the effects of tension on the kinetics of DNA looping, we used optical tweezers in conjunction with tethered-particle motion (TPM) measurements to inves- tigate the formation and breakdown of LacI-mediated DNA loops under a constant stretching force. We report three main results: First, the rate of loop formation is extremely sensitive to applied tension resulting in a tenfold decrease in loop formation when increasing the tension from 60 to 183 fN. Second, the lifetime of the looped state appears to be completely 2