Objective Medical Research cannot be separated from miniature pigs animal models. Due to the special anatomical structure of the oropharynx and the glottis is difficult to be exposured by laryngeal, the establishment of the airway is the most critical step in the perioperative period of miniature pigs. How to perform intubation while preserves spontaneous breathing has been one of the hot spots in the experimental research field of miniature pigs. This study intends to investigate the effect of small-dose Zoletil combined isoflurane inhalation in preserved spontaneous breathing endotracheal intubation of miniature pigs and to explore the minimum alveolar effective inhaled concentrations in 50% (MAC EI50) and minimum alveolar effective inhaled concentrations in 95% (MAC EI95) to provide a reference for clinical practice. Methods 26 miniature pigs were plan to be operated under general anesthesia. Anesthesia was conducted by Zoletil intramuscular?inject and isoflurane inhalation. Heart rate, blood pressure, respiration rate, body temperature, oxygen saturation, end-tidal CO2 partial pressure and end-tidal isoflurane concentration were monitored. The initial end-tidal isoflurane concentration was set 2.0% isoflurane increased or decreased according to a modified sequential method with an adjacent concentration ratio of 1.1. Tracheal intubation conditions and efficacy were evaluated by Cooper's score. MAC EI50, MAC EI95 and corresponding 95% CI of alveolar isoflurane concentration during endotracheal intubation with direct laryngoscope was calculated by Probit regression. Results The MAC EI50 of alveolar isoflurane concentration in miniature pigs during endotracheal intubation with direct laryngoscope was 3.07% (95% CI 2.70-3.61%), MAC EI95 was 3.82% (95% CI 3.41-7.49%). Conclusion Small-dose Zoletil combined with isoflurane inhalation is a better and safe anesthesia method for preserved spontaneous breathing endotracheal intubation in miniature pigs. The MAC EI50 of alveolar isoflurane concentration in miniature pigs during endotracheal intubation with direct laryngoscope was 3.07% (95% CI 2.70-3.61%), MAC EI95 was 3.82% (95% CI 3.41-7.49%).