Disclosed is a L-isoleucine-producing Corynebacterium glutamicum fermentation medium, comprising a basal medium and a growth factor, wherein the growth factor consists of choline, betaine and vitamin B6, and the contents of each ingredient in the fermentation medium are: 0.2-1 g/L choline, 0.25-0.5 mg/L betaine, and 0.05-0.3 mg/L vitamin B6. Also disclosed is a method for cultivating the L-isoleucine-producing Corynebacterium glutamicum, comprising: inoculating the L-isoleucine-producing Corynebacterium glutamicum onto the fermentation medium, wherein the volume of the bacteria liquid accounts for 5-20% of the volume of the fermentation medium, adjusting the pH to 6.5-7 with aqueous ammonia, controlling the dissolved oxygen to 30-50%, and fermenting for 25-30 h; then decreasing the dissolved oxygen to 15-25%, and feeding a 50-80% glucose solution into the fermentation broth to control the residual sugar at 3-4%, continuing the fermentation until 60-70 hours, then terminating the fermentation, and controlling the temperature of the overall fermentation process at 29-33° C.