We investigated the cell viability, proliferative capacity and neuronal differentiation potential of canine bone marrow stromal cells (BMSCs) after cryopreservation. BMSCs were cryopreserved using cryoprotectant solutions with 10% DMSO and 10% FBS (DF group) or without DMSO and FBS (DF-free group); fresh BMSCs were used as a control. The cell viability and proliferative capacity of BMSCs were similar in the DF-free and control groups, while those in the DF group were lower. In all groups, BMSCs differentiated into neuron-like cells that stained positive against neuron markers, and the mRNA expression levels of neuron markers increased after neuronal induction. In conclusion, cryopreservation with DF-free cryoprotectant solution did not diminish the cell viability, proliferative capacity or neuronal differentiation potential of canine BMSCs.