Context Black-tailed prairie dogs (Cynomus ludovicianus) often come into conflict with humans in urban natural areas adjacent to private property by destroying plants and denuding the landscape. There is a diversity of attitudes related to urban prairie dog management in Colorado and interest in non-lethal methods is increasing. Therefore, the development of non-lethal methods that allow the existence of prairie dogs and mitigate damage related to population density is needed. Aims The efficacy of DiazaCon (20,25-diazacholesterol dihydrochloride, a cholesterol analogue) as an oral contraceptive bait for reducing reproduction in black-tailed prairie dogs was tested. Persistence of DiazaCon in black-tailed prairie dogs 11 months after initial application was also determined. Methods Prairie dogs were baited with DiazaCon-coated rolled oats or control bait for 10 days over a 20-day period in November 2007 on one control and one treatment site. Sites were randomly assigned to a treatment group. Visual counts were used 8 months post-treatment (June/July 2008) to determine pup : adult ratios on both sites. Prairie dogs on both sites were trapped 11 months post-treatment (September/October 2008) to gather mass and age data to assess juvenile : adult ratios and to collect blood samples for determination of serum cholesterol and desmosterol concentrations. Key results DiazaCon treatment reduced the number of pups per adult by 95.5% when compared with the control site in June/July 2008 (0.09 ± 0.02, n = 10, number of observation days) versus 2.0 ± 0.2 (n = 10). Free serum cholesterol levels in treated animals (370.7 µg mL–1) were lower than control animal levels (411.6 µg mL–1, P < 0.015), while treated desmosterol levels (14.5 µg mL–1) were higher than controls (0.35 µg mL–1, P < 0.02) 11 months after ingestion of DiazaCon bait. However, these differences would likely not have been sufficient to prevent successful breeding in the upcoming breeding season. Conclusions DiazaCon is an effective contraceptive in black-tailed prairie dogs. It is probably reversible and will likely affect breeding success for only one breeding season. DiazaCon may provide an additional tool to help manage urban populations of prairie dogs in locations where lethal control is unacceptable. Implications The need for further research into secondary hazards to non-target animals, such as predators, is discussed. Recommendations for field application are also discussed.