: Non-surgical neutering strategies have long been pursued for male animals. A previous study from our group showed that magnetic nanoparticle hyperthermia (MNH) applied directly to the testicles is a promising non-surgical sterilization method for male animals, causing testicular atrophy and complete disappearance of seminiferous tubules by the end of a 56 day observation. This long-term study was conducted to verify the long-term efficacy and safety of the method. : Wistar rats treated with testicular MNH received an intratesticular injection of a magnetic fluid composed of manganese-ferrite nanoparticles functionalized with citrate (MnFeO-citrate) and were subsequently subjected to an alternating magnetic field. Reproductive parameters and animal health were evaluated by blood tests and abdominal ultrasound for 12 months. : All MNH-treated animals presented testicular degeneration and atrophy, together with severely reduced or undetectable serum testosterone levels. By the end of the experiment, all but two animals had no identifiable gonads. The only two animals still displaying gonadal-like structures were azoospermic, and histopathology revealed the remaining tissue was non-functional. The procedure was well-tolerated and MNH-treated animals presented no long-term side effects. Hemogram, ALT, AST, urea and creatinine levels were within the normal parameters for Wistar rats over the 12 month period. The liver, spleen, kidneys and lungs had normal structures as revealed by abdominal ultrasound and histopathological exams, with no nanoparticle accumulation in the organs over the long term. : In conclusion, testicular MNH caused irreversible infertility in rats in a single application, with no adverse effects on general animal health.