Objectives: This study is aimed at evaluating gender differences in neural activity change response to the acupuncture on left Jianyu (LI 15) in healthy volunteers.Methods: Forty healthy volunteers (20 males and 20 females) received 20‐min acupuncture on left LI 15 and underwent functional magnetic resonance imaging (fMRI) scans before and after acupuncture. Amplitude of low‐frequency fluctuations (ALFF) in the 0.01–0.08 Hz range were determined for both scans. Paired t‐tests were performed on ALFF between two scans separately for the male and female groups to identify neural changes related to acupuncture.Results: After acupuncture, males showed significantly increased ALFF in the left cerebellum and right angular gyrus but decreased ALFF in the left precentral gyrus, left inferior occipital gyrus, and right fusiform gyrus. However, the ALFF change in females is almost negligible.Conclusions: Brain functional activity in response to acupuncture on left LI 15 is noticeably different between males and females. This is preliminary evidence that gender may be an important factor for optimal clinically personalized acupuncture therapy for poststroke shoulder pain in the future.