Background In ambulance care, patients are often classified as high-risk, particularly when they are in immediate need of pain relief. It has been shown that, after ambulance nurses administer intravenous analgesic drugs, patients delivered to the emergency department tend to complain of moderate to severe pain. Aims and objectives The present study aimed to evaluate the overall patient-perceived pain during treatment with methoxyflurane (MTX) in an ambulance-care setting. We also explored potential demographic variations. Methods This prospective observational study included 50 patients in need of ambulance care that perceived acute pain, defined as a Numeric Rating Scale (NRS) value â¥4 (scale range: 1â10). We monitored the vital parameters of patients and MTX treatment characteristics, including the total number of inhaled MTX breaths and the average number of treatment sequences. Results Among the 50 patients initially assessed, we excluded 8 patients (16%), due to MTX contraindications. We excluded 10 patients (24%), due to discontinued treatment. The remaining cohort (nâ¯=â¯32) that fulfilled the pain-relieving procedure, included equal numbers of men and women. The average time spent in ambulance care was 29â¯Â±â¯15â¯min. The NRS scores for pain measured at the scene (median 8.0, interquartile range [IQR]: 7.25â10.0) were significantly higher than those measured at hospital admission (median 5.0, IQR: 4.0â¯7.0; pâ¯=â¯.001). The median NRS scores measured at the hospital were different between sexes (men: 6.0, IQR: 5â7.25; women: 4.0, IQR: 3.76â6.0; pâ¯=â¯.036). The average number of treatment sequences was 2. The overall average number of inhaled breaths was 17â¯Â±â¯9. Conclusion This study demonstrates that MTX provided clinically significant lower pain scores among patients in ambulance care without significant effects on vital parameters. However, the pharmacological characteristics of MTX appeared to affect the potential of achieving standardized treatment objectives.