Aims As cell-adapted foot-and-mouth disease virus (FMDV) with H56R mutation in VP3 has reduced thermostability, this study aimed to investigate the effect of thermostabilizers on cell-adapted FMDV for vaccine production. Methods and Results We examined the effect of 3% sucrose, 10% (or 25%) glycerol or 10% FBS on cell-adapted FMDV O/SKR/JC/2014, containing H56R mutation in VP3, as vaccine seed virus at -80, 4, 25 or 37 degrees C for 2, 4 or 7 days. The stabilizing effect of 3% sucrose on O/SKR/JC/2014 was observed at 25, 37 degrees C, and after repeated freeze-thaw cycles. Additionally, we tested the effect of 3% sucrose on the growth of FMDV or cells and did not observe any decrease in either viral growth or cell viability. Conclusions Our study showed the protective effect of 3% sucrose on FMDV infectivity at various temperatures; this virus stock in 3% sucrose could be used for infecting cells without the removal of sucrose. Significance and Impact of the Study We suggest that 3% sucrose-containing medium could be beneficial for the stable storage and transport of cell-adapted FMDV.