In recent years, the incidence of cosmetic injection-induced botulism has remarkably increased due to the frequent usage of botulinum neurotoxin type A (BoNT/A). To mimic and investigate this new clinical type of botulism, we established a novel animal model and evaluated the therapeutic potential of a new drug. Firstly, we injected BoNT/A into the gastrocnemius of rats to induce partial paralysis of the remaining limbs. Then, the intoxicated rats were treated with 3,4-diaminopyridine (3,4-DAP) at different stages of the disease and the forelimbs grasping strength (FGS) was evaluated. We showed that, at the sublethal dose, the FGS began to decrease at 6.00 ± 1.86 h after injection in rats, from 2.28 ± 0.19 N to 1.51 ± 0.18 N, while the FGS declined appeared earlier (4.29 ± 0.42 h) at the lethal dose, from 2.30 ± 0.20 N to 1.20 ± 0.16 N. Treatment with 3,4-DAP respectively at the time of the symptoms onset or 7 days after injection both can temporarily reverse the symptoms of muscle paralysis, indicating that 3,4-DAP may be an effective approach to relieve botulism. Overall, this study provides an available rat model and a promising therapeutic strategy for cosmetic injection-induced botulism.