The precise timing required for successful fixed-time artificial insemination (AI) can be a logistical challenge for producers with large, diverse herds of cattle. A novel method of bull sperm storage at ambient temperature has been developed to avoid the damaging freeze/thaw process, with the aim of extending the post-insemination lifespan of sperm in the female reproductive tract. Previously, this novel method of ambient temperature sperm storage in SpermSafe-B has shown promising results when fixed-time AI using 7-day stored sperm was performed on 2-year-old virgin Bos taurus heifers (n=18; 14 of 18 pregnant). To assess the fertility of Bos indicus sperm in this medium, a herd of 12 heifers (9 Bos indicus, 1 taurine-indicine cross, and 2 full Bos taurus) underwent a 12-day oestrus synchronisation protocol and were each inseminated with approximately 25 million 7-day stored Bos indicus sperm. At least 67% (8/12) of the heifers are known to have been cycling and all had body condition scores between 2 and 3. At a 35-day ultrasonographic pregnancy diagnosis, six of the 12 heifers (50%) were confirmed pregnant. These findings demonstrate that the fertility of Bos indicus sperm can be maintained in SpermSafe-B for 7 days of storage at room temperature and that these stored sperm have the capacity to achieve pregnancy in Bos indicus females. Ambient temperature sperm storage using SpermSafe-B could be a viable alternative for Bos indicus breeds, as well as Bos taurus breeds, when AIs will be performed within 7 days of semen collection. Although viability and motility have been maintained beyond 7 days of storage, there is a significant increase in viable sperm with destabilised membranes between days 10 and 14 (%M540 positive live cells: 19.4±5.70% vs. 47.4±7.64%; P≤0.005), suggesting 10 days may be the maximum duration of fertility for sperm stored in SpermSafe-B.