Objective To evaluate the anesthetic effect and safety of premedication midazolam combined with butorphanol on isoflurane-anesthetized mice. Methods Seventy five mice were randomly divided into three groups of 25 mice: control group(receiving isoflurane anesthesia only), observation group(receiving premedication midazolam combined with butorphanol with isoflurane anesthesia)and blank group(receiving no anesthesia). The indices of heart rate(HR) and oxygen saturation(SpO₂ )were assessed at induction and every 5 min post-induction for 1 h. The serum levels of glucose (Glu)and cortisol(Cor)were measured and compared pre- and post-induction. The indices of induction and recovery time, and analgesic and sedative time were measured and compared among the three groups. Adverse reactions during induction were also recorded. Results Compared with the control group, the indices of induction time, sedation onset time and pain- loss time were shorter in the observation group(P<0. 05), but the recovery time was no different(P>0. 05). The HR index in the blank group was the highest among the three groups(P<0. 05). The HR index of the control group was higher than that of the observation group during 5~ 35 min post-induction(P<0. 05), and the HR index of the control group was lower than that of the observation group during 35~ 60 min post-induction(P<0. 05). The SpO₂ index of the control group was the lowest among the three groups after 1 h post-induction ( P < 0. 05), and the SpO₂ index was not different between the observation and blank groups(P>0. 05). The HR variation tendency was different among the three groups(F= 15. 215, P= 0. 000). The SpO₂ variation tendency was not different among the three groups(F= 1. 015, P= 0. 180). The serum levels of Glu and Cor were not different among the three groups pre-anesthesia(all P>0. 05). The serum levels of Glu and Cor in the control group were the highest among the three groups at mid-anesthesia(all P<0. 05), but no difference was found between the blank and observation groups at mid-anesthesia(all P>0. 05). The serum levels of Glu and Cor at mid-anesthesia were higher than at pre-anesthesia in the control group ( P< 0. 05), but the serum levels of Glu and Cor in the blank and observation groups were not different between pre-anesthesia and mid-anesthesia ( P> 0. 05). The incidences of adverse reactions were not different between control and observation groups(P= 0. 626). Conclusions The anesthetic effects of premedication midazolam combined with butorphanol on isoflurane-anesthetized mice are safe and reduce the degree of respiratory inhibition.