Follicle-stimulating hormone (FSH) must be applied at 12-h intervals over 4–5 days in the traditional cattle superovulation protocol, which still needs to be improved. This research paper evaluated the superovulation results obtained by a traditional protocol or by a single administration of FSH dissolved in MontanideTM ISA-206 VG (MonISA-206). Control cows were superovulated with 10 mL of FSH (500 µg pFSH + 100 µg pLH) from day 7 to day 10 (for 4 days, twice daily i.m. injections, decreasing doses). Cows in the EG10 and EG7.5 groups were injected i.m. with 20 mL (100%, 10 mL + 10 mL) or 15 mL (75%, 7.5 mL + 7.5 mL) of the FSH and MonISA-206 mixture at once on day 7. All cows were inseminated 12 and 24 h after oestrus onset. The cows presented no pathology at the injection sites. Plasma FSH levels differed between the groups, but the interaction between hour and group × time was not different. Superstimulation and embryo quality results were similar between the groups. A single injection of FSH (both 100% and 75% doses) dissolved in MonISA-206 led to adequate plasma FSH levels and similar superovulation results to traditional FSH treatment, and caused no pathology at the injection sites.