In relations to cold acclimation, experiment was carried out to understand the seasonal changes in reserve substances of the mulberry. The shoot barks and leaves of three mulberry varieties(Kaeryangppong, Shinilppong and Yongcheonppong) were sampled, after that their reserve substances were analyzed. The cold hardiness of mulberry was investigated by DTA(Differential Thermal Analysis) method. To increase cold hardiness, gibberellin(100 ppm), kinetin(100 ppm) and Jambi 8 were sprayed on the mulberry leaves. After spraying, falling of the leaves of Yongcheonppong occured earier than the other varities. After the first frost, all of treatments except gibberellin were entirely fallen. Growth regulator extended the leaves fallen. After spraying, water of the shoot barks was not showed difference in the content among the treatments, but amino acid, carbohydrate and soluble protein increased from September to October. Starch content of the shoot barks and leaves was maximum in October, but thereafter decreased during wintering stage. In Shinilppong, Jambi 8 spray increased cold hardiness by 1-2$^{circ}C$ more than no spray. It was concluded that the cold hardiness of the mulberry in midwinter is closely related to the reserve substances with spraying Jambi 8 on the mulberry leaves.