Background Although a certain level of competitive anxiety may increase performance, many athletes with anxiety experience uncontrolled negative feelings and cognition that in turn can have overwhelming effects on their performance. Methods We aimed to assess the effect of acupuncture on competitive anxiety of the adolescent football players prior to the competition using psychological and physiological markers. A total of 30 athletes were randomly and equally allocated to either acupuncture or sham control group. Results The results of t-test on posttest scores showed that acupuncture had a significant effect on cognitive anxiety (p=0.001) and somatic anxiety (p<0.001) but not self-confidence (p>0.05). Furthermore, the results showed that acupuncture significantly decreased the skin conductance in acupuncture group compared to sham group (p=0.006) (p<0.001). Conclusions In conclusion, the results suggested that acupuncture has the capacity to decrease cognitive and somatic anxiety prior to competition in adolescent athletes while this was accompanied by significant physiological changes.