The purpose of this study was to investigate the effect of vitamin B2 deficiency on the utilization and recuperation of fuel in exercising rats. Thirthy six rats were fed either a vitamin B2 deficient diet (-B2) or a control diet (+B2) for 3 weeks and then subdivided into 3 groups : non-exercise group(NE), exercise group (ES), exercise and recuperation group (ER). ES group were exercised on treadmill (10$^{circ}$, 0.5-0.8km/h) for 2 hours and ER group were recuperated three days with the respective diet after exercise. Glucose (GLU), glycogen(GLY), protein(PRO), triglyceride(TG) and free fatty acid(FFA) were compared in plasma(P), liver(L) and skeletal muscle(M) of rats. Compared to + B2 rats, in NE group, the level of P-GLU and L-GLU of -B2 rats was lower, L-PRO and M-GLY was higher and there were no differences in P-PRO, P-FFA, L-TG and M-TG. In ES group, the level of P-GLU was lower, P-FFA was higher and there were no differences in P-PRO, P-TG, L-GLU, L-TG, M-GLY, M-TG and M-PRO. In ER groups, the level of P-GLU and L-TG was lower, P-FFA was higher and there were no differences in P-PRO, P-TG, L-GLY, L-PRO, M-GLY, M-TG and M-PRO. These results suggest that a vitamin B2 deficiency may impair the utilization of stored fuel during exercise suggest that a vitamin B2 deficiency may impair the utilization of stored fuel during exercise and may lead a sluggish recuperation related to fuel stores after exercise.