This study aimed to describe spinal cord injured patients’ semen characteristics before and after cryopreservation, and assisted reproductive technology cycles outcome compared to the infertile population. Data about sperm analysis and assisted reproductive technology (ART) cycles outcomes for 78 men with spinal cord injury referred for sperm cryopreservation between 1998 and 2013 were retrospectively analysed and compared with a reference group consisting of every Intra Cytoplasmic Sperm Injection (ICSI) cycle performed in our in vitro fertilization unit over the 2009–2014 period. Semen was collected by penile vibratory stimulation (PVS), electroejaculation or via testis biopsies. Fresh and frozen semen parameters and ART outcomes with frozen-thawed sperm were measured. Patients were divided into three groups according to the sperm retrieval methods: 37 electroejaculations, 37 PVSs and four surgical sperm retrievals. Low ejaculate volume was observed in 33% of the patients, and oligozoospermia in 37% of the patients. Specimens from 77 of the patients contained motile sperm and were therefore frozen for future use. There was no statistical difference for any of the fresh semen parameters between all groups. Twenty of them underwent ICSI leading to five live births, while four underwent intrauterine insemination. ART with frozen-thawed spermatozoa can provide men with SCI with comparable results as in the infertile population.