Urethane is widely used for its ability to induce prolonged anesthesia. Variability in previously reported cardiovascular parameters in murine models makes it challenging to definitively evaluate the cardiovascular effects of urethane anesthesia. We aimed to address these challenges, thereby advancing our understanding of urethane's effects on cardiovascular function in mice. In this study, we investigated how urethane anesthesia, with and without supplemental oxygen, impacts heart rate, arterial oxygen saturation ([Formula: see text]), blood pressure, and heart rate variability in mice. First, we conducted a literature review and found that data in mice were both limited and lacking in reproduction. Next, we conducted a series of physiological measurements to address gaps in the literature and subjected C57BL/6J mice to three conditions: ) conscious, ) urethane-anesthetized, and ) urethane-anesthetized with supplemental oxygen. Blood pressure, heart rate, [Formula: see text], and heart rate variability (via time, frequency, and M-curve analyses) were assessed. We observed an increase in heart rate with urethane anesthesia ( = 0.012) compared with the conscious state. Urethane caused a decrease in heart rate variability, which was independent of oxygen supplementation. Urethane anesthesia caused a significant reduction in arterial blood pressure ( < 0.001) with oxygen-supplemented mice remained hypotensive. Urethane decreased [Formula: see text] ( = 0.001), which was restored by oxygen supplementation ( < 0.001). We did not observe sex effects of urethane anesthesia on blood pressure, heart rate, heart rate variability, or [Formula: see text]. Taken together, these results underscore the importance of a cautious approach when using urethane in mice, as urethane significantly impacts arterial blood pressure, heart rate, oxygen saturation, and heart rate variability. This investigation documents cardiovascular outcomes in mice receiving urethane anesthesia, emphasizing sex as a biological variable, and considering oxygen supplementation during anesthesia. This is the first report of M-curve analysis in rodents as a heart rate-independent variability analysis.