The endangered black-footed ferret (BFF; Mustela nigripes) is an important example of the benefits of assisted reproduction in species conservation with both semen evaluation and artificial insemination using fresh and frozen sperm being successfully incorporated into the breeding program. Currently, electroejaculation (EE) is routinely utilized for semen collection in BFFs, a technique that requires custom equipment and experienced operators, and does not consistently yield viable samples in this species. In this case study, we evaluated the feasibility of urethral catheterization (UC) for semen collection, a method predominately tested in domestic and non-domestic felids, on four occasions (three BFF males). After general anesthesia with a combination of ketamine, midazolam and α2-agonist dexmedetomidine (thought to promote semen release into the urethra), a lightly lubricated, flexible feeding tube was passed into the urethral opening and advanced ~7–8 cm into the urethra. A syringe attached to the feeding tube was used to apply mild negative pressure to collect sperm. Semen samples were successfully collected on all four attempts. Sperm characteristics ranged as follows: 10.5–26.0 × 106 sperm/ml concentration, 50–90% motility and 36–61% normal sperm morphology. This is the first report of the use of UC as a potential alternative to EE in the BFF, a more field-friendly technique that is less invasive and more consistent for obtaining samples free of urine contamination.