Protection of cells from osmotic stress is crucial for their survival. Exposure to high osmolarity promotes rapid diffusion of water across cell membranes, dramatically increasing cellular ionic strength, leading to disruption of key proteins/DNA resulting in cell-cycle arrest and apoptosis. The luminal microenvironment of the epididymis is hypertonic; therefore, epididymal cells adapt to the higher osmolarity by accumulating organic osmolytes, such as L-carnitine. Osmolytes do not perturb cells when accumulated in high concentrations, nor do they affect key proteins or damage DNA. Therefore, osmolytes and their transporters are crucial for cell survival. Transporters that are responsible for the accumulation of organic osmolytes have been shown to be regulated at the transcriptional level by hypertonicity. The present study examines the gene expression of known osmoprotective/stress genes in epididymal cells exposed to changes in tonicity. We demonstrate that the osmoprotective/stress pathways present in other organs, such as the kidney, operate in the epididymis, potentially aiding in the protection of its luminal cells and spermatozoa. Further, it was also seen that OCTN2, a transporter that is thought to be responsible for the accumulation of L-carnitine in the epididymal lumen, is regulated in response to changes in tonicity.