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Simple Summary: Turtles and tortoises are routinely presented to veterinarians for medical and surgical treatment. In many of these cases, sedation is required to properly examine the animal and perform necessary diagnostic tests. Unfortunately, there is a dearth of evidence-based research available to guide veterinarians working with these animals. In addition, the available publications show that there are differences between species. Therefore, with nearly 350 species of chelonians in the world, it is important for us to evaluate protocols on a species-by-species basis. In this study, six eastern mud turtles (Kinosternon subrubrum) were given 10 mg/kg alfaxalone via intramuscular (IM) and intravenous (IV) routes using a cross-over study design that allowed us to compare the results between and within turtles. As expected, the IV route led to a faster induction and recovery. This study demonstrated that alfaxalone 10 mg/kg IV or IM can be used to provide safe and effective sedation in eastern mud turtles. Current sedation protocols for chelonians can pose a challenge to clinicians because of prolonged induction and recovery times, difficulties in gaining venous access, and natural species variation. This study evaluated the sedative and physiologic effects of intramuscular (IM) and intravenous (IV) alfaxalone in six wild-caught adult eastern mud turtles (Kinosternon subrubrum). The turtles received alfaxalone 10 mg/kg IM and IV in a randomized cross-over design. A 10-day washout period occurred between trials. Baseline parameters (heart rate, respiratory rate, temperature, and reflexes) were assessed prior to injection and every 5 min post-injection until recovery. Three venous blood gas samples were also collected and analyzed over the course of each trial (baseline, induction, and recovery). Intravenous alfaxalone resulted in a significantly faster induction (p = 0.016; median: 1.5 min, 25–75%: 1–7.5, minimum–maximum: 1–21) and a shorter total sedation time (p = 0.041; median: 52 min, 25–75%: 34.5–62.5, minimum–maximum: 33–87) when compared with IM alfaxalone (induction, median: 20 min, 25–75%: 15–22.5, minimum–maximum: 15–25; total, median: 70 min, 25–75%: 65–82.5, minimum–maximum: 65–90). Blood gas and physiologic parameters were not significantly different between groups; however, the pH (p = 0.009) and glucose (p = 0.0001) significantly increased, and partial pressure of carbon dioxide (p = 0.024) significantly decreased over time. This study demonstrated that alfaxalone 10 mg/kg IV or IM can be used to provide safe and effective sedation in eastern mud turtles. [ABSTRACT FROM AUTHOR]