Objective To manufacture a simple device for securing the hind limbs of mice for acupuncture and moxibustion treatment, and provide a convenient economical, efficient, and practical tool for acupuncture and moxibustion experiments. Methods A holding device for mouse hind limbs was made according to design drawings. Four investigators secured mice with specialized cloth ( clothing method ) or the mouse hind limb device, and then needled mice at the Zusanli point. To compare the effects of these two holding method in acupuncture, the holding time, number of hind limbs retracted, number of needles shed and the body weight of mice were recorded, and the influence on the stress response of mice observed. In addition, the effects of different holding method on pain threshold, cognition and emotion were observed by the thermal radiation pain threshold, open field and elevated cross maze tests. Results Compared with the clothing method , there was no significant difference in the body weight of mice with the holding device during acupuncture. The mouse hind limb holding device was easy to make, more efficient, and had less frequent hind limb retraction and needle shedding compared with the clothing method. There were no significant differences in the thermal radiation pain threshold, open field and elevated cross maze tests between the two holding method. Conclusions The hind limb holding device was convenient, which helped to improve the efficiency of the experiment, and had no significant impact on pain threshold, cognition and emotion compared with the clothing method. The holding device can be used for specific experimental needs.