Background and objective <p>Most troubling of painless gastrointestinal endoscopy is hypoxia and hypotension. Esketamine has sympathomimetic and respiratory protection, which combined with midazolam and propofol may reduce these troubles. So, this study aims to explore the efficacy and safety of this regimen.</p> Methods <p>In total, 318 patients who received painless gastrointestinal endoscopy from November 2022 to April 2023 were included and randomly divided into two groups at 1:1: 0.1 µg/kg sufentanil (Suf) or 0.15&#xa0;mg/kg esketamine (Esk) group. Except for analgesics, all patients were given midazolam and propofol. The incidence of hypotension and hypoxia (SPO₂ &lt; 93%) during the examination was observed.</p> Results <p>Compared with the Suf, the Esk group reduced the incidence of hypotension (12.8% vs 36.2%, 95% confidence interval (CI) 13.20—33.09, <i>P</i> &lt; 0.001), respiratory depression (apnea &gt; 30&#xa0;s) (2.1% vs 15.4%, 6.47—20.52, <i>P</i> &lt; 0.001), and hypoxia (7.1% vs 16.1%, 1.07—16.92, <i>P</i> = 0.018), but may increase the incidence of coughing (<i>P</i> &lt; 0.001) and increased secretions (<i>P</i> = 0.032). There was no significant difference in the examination and recovery time (<i>P</i> &gt; 0.05).</p> Conclusion <p>Esketamine, midazolam, and propofol for painless gastrointestinal endoscopy can achieve opioid-free sedation and reduce the occurrence of hypotension and hypoxia. However, the dosages of this regimen need to be further explored to avoid coughing and increased secretions.</p> Trial registration <p>This study has been registered in the Chinese Clinical Trial Center (<a href="https://www.chictr.org.cn/index.html">https://www.chictr.org.cn/index.html</a>, Date: November 3, 2022,&#xa0;No: ChiCTR2200065379).</p>