Artificial Insemination (AI) is underutilized by northern Australian beef cattle producers due to the logistical complications associated with using chilled or cryopreserved sperm which requires oestrus synchronization and precise timing of insemination. Through the development of an ambient temperature sperm preservation medium, the lipid membrane phase changes that occur during cooling can be avoided so that spermatozoa will maintain greater longevity and fertility after insemination. The aim of this study was to develop an ambient temperature sperm storage medium to maintain sperm motility and viability for at least one week. As the regulation of intracellular sodium presents a major source of ATP loss during cell storage, the effect of replacing sodium chloride with L-carnitine as the primary osmolyte in a modified Biggers, Whitten, and Whittingham (BWW) medium was investigated. Bull semen was collected by electroejaculation (N=8) and viable spermatozoa were selected using a density gradient. When compared to spermatozoa stored at 5°C in a commercial semen extender (INRA96), the carnitine-BWW (at RT) had significantly higher total (TM) and progressive (PM) motilities at day 3 (TM: 71.63±3.85% vs. 58.75±6.50%; PM: 38.00±4.10% vs. 11.50±2.73%; P≤0.0015 and <0.0001 respectively), 7 (TM: 65.88±4.76% vs. 51.25±6.17%; PM: 27.25±5.18% vs. 9.50±3.39%; P≤0.0005 and <0.0001), and 10 (TM: 59.88±4.25% vs. 35.25±8.14%; PM: 19.00±4.18% vs. 5.50±2.41%; P≤0.0008 and ≤0.0032) and significantly higher viability (53.88±3.44% vs. 25.18±3.14%; P<0.0001) and acrosome integrity (41.34±4.34% vs. 23.66±3.03%; P<0.0001) after 3 days. Spermatozoa is stored at low temperatures to reduce its metabolic rate. At higher temperatures, the uncurbed metabolic activity results in high levels of reactive oxygen species (ROS) and depletion of ATP. The results of this study show that L-carnitine is a potent pro-survival factor for bull spermatozoa, most likely through its actions as an osmolyte and a powerful antioxidant, along with its role in mitochondrial ATP synthesis.