The combination of botulinum toxin type A (BoNT/A) and energy equipment have been widely used in the clinic.To determine whether the energy of microneedle fractional radiofrequency (MFR) affects the efficacy of BoNT/A and to provide an optimal strategy for the energy device in combination with BoNT/A in the clinic.First, a total of 45 females with moderate-to-severe periorbital crow's feet wrinkles were enrolled and divided into three groups according to different treatment methods and intervals, including BoNT/A injection alone, BoNT/A injected immediately after MFR treatment and BoNT/A injected 7 days after MFR treatment. The photographs were compared before treatment and 4 weeks after treatment. Then, the mouse models were established by combining MFR with BoNT/A at different intervals, to evaluate muscle strength, muscle mass, muscle nutritional markers, and important cytokines levels.All patients in each group had high satisfaction. The MFR + BoNT/A (immediately) group could improve dynamic wrinkles, but the others had more significant efficacy (p < 0.05). The results of mouse models showed that all BoNT/A groups induced different degrees of muscle paralysis in vivo, but the paralytic effect induced by the BoNT/A group, MFR + BoNT/A (interval of 3-day) group, and MFR + BoNT/A (interval of 7-day) group were higher than others and the expression levels of muscle nutritional markers in NMJ tissues were significantly upregulated.MFR has a certain reduction effect on the activity of BoNT/A, and this reduction effect would last for 3 days after MFR treatment.