The intradiscal application of mesenchymal stem cells (MSCs) is considered a promising strategy for intervertebral disc (IVD) regeneration. Although many studies have been published, the feasibility and regenerative effects of intradiscal MSC application have not been evaluated in an animal model suffering from naturally occurring degenerative disc disease. Six German Shepherd Dogs suffering from naturally occurring degenerative IVD disease were included. Autologous MSCs were isolated from bone marrow (iliac crest) and cultured for 3 weeks. After decompressive spinal surgery, three dogs received an intradiscal injection of MSCs, while the other three dogs received an intradiscal injection of saline (control). Clinical status, disc height index, Pfirrmann grading, and disc volumetry were evaluated at 1, 6, and 12 months after treatment. Autologous application of canine MSCs was feasible and successful in all dogs. No evident complication was found. Surgery resulted in an equal improvement in clinical status in the treatment and control dogs. In the MSC group, the Pfirrmann grade increased in all patients over time, whereas in the control group, the Pfirrmann grade remained stable. The volume of the L7-S1 IVD gradually increased during the 12-month study period in all dogs, with no evident difference between the MSC and control group. On the basis of this preliminary study, it can be concluded that intradiscal injection of autologous MSCs in dogs with spontaneous degenerative IVD disease is well tolerated without any adverse effects, does not affect clinical outcome, and does not have any evident regenerative effects.