Successful cryopreservation of bison semen is fundamental for restoration of genetic diversity in Canada's wood bison. Conventional bovine semen extenders contain animal products, such as egg yolk and milk, which are undesirable because of biosecurity risks and undefined composition. In this study, we examined the efficacy of an exogenous protein-free extender containing cholesterol-cyclodextrin complex (CC) to cryopreserve bison semen. The study also provided an opportunity to determine the effectiveness of different ovulation synchronization protocols for fixed-time artificial insemination in bison. Semen was collected from wood bison bulls via electroejaculation and cryopreserved in either Tris-egg yolk-glycerol (called ‘TEYG’) extender or pretreated with CC (2 mg/mL semen) and diluted in Tris-glycerol (collectively called ‘CC-TG’) extender. Post-thaw sperm motion characteristics and in vitro fertilization of cattle oocytes confirmed that CC alone without egg yolk protected bison sperm during cryopreservation process. In the first fertility trial, however, no pregnancy was obtained following fixed-time artificial insemination of bison cows with CC-TG extender. In a follow-up trial, low concentration of CC (1 mg/mL semen) resulted in better post-thaw sperm motion characteristics, fertility rate, and birth of live calves following fixed-time artificial insemination. Results showed that 1 mg CC/mL semen completely replaced egg yolk in bison semen extender. In addition, both follicular ablation and steroid treatment protocols provided ovulation synchrony to permit successful application of fixed-time artificial insemination in bison. This is the first report on the birth of live bison calves following fixed-time artificial insemination using semen cryopreserved in a defined extender.