Lumbar puncture (LP) is stressful and often painful. We evaluated the efficacy of a fixed 50% nitrous oxide–oxygen mixture (50%N2O-O2) versus placebo to reduce immediate procedural pain and anxiety during LP performed in an emergency setting. We conducted a randomized controlled trial involving adults who needed a cerebrospinal fluid analysis in an emergency department. Patients were randomly assigned to inhale either 50%N2O-O2 or medical air. The primary endpoint, assessed using a numerical scale, was the maximum pain felt by the patient during the procedure and the maximum anxiety and satisfaction as secondary outcomes. Eighty-eight patients were randomized and analyzed (ITT). The maximal pain was 5.0 ± 2.9 for patients receiving air and 4.2 ± 3.0 for patients receiving 50%N2O-O2 (effect-size = −0.27 [−0.69; 0.14], p = 0.20). LP-induced anxiety was 4.7 ± 2.8 vs. 3.7 ± 3.7 (p = 0.13), and the proportion of patients with significant anxiety (score ≥ 4/10) was 72.7% vs. 50.0% (p = 0.03). Overall satisfaction was higher among patients receiving 50%N2O-O2 (7.4 ± 2.4 vs. 8.9 ± 1.6, p < 0.001). No serious adverse events were attributable to 50%N2O-O2 inhalation. Although inhalation of 50%N2O-O2 failed to reduce LP-induced pain in an emergency setting, it tended to reduce anxiety and significantly increased patient satisfaction.