Anesthesia can affect measured thyroxine (total T4) concentrations in humans and animals, but its effect in black-tailed prairie dogs (Cynomys ludovicianus) has not yet been studied. We used isoflurane to anesthetize 12 prairie dogs for 60 min. Blood samples were obtained from each animal immediately after anesthesia induction and at 30 and 60 min and used for analysis of plasma T4 concentration. The plasma T4 concentration (mean ± 1 SD) was significantly decreased from baseline (3.49 ± 0.52 μg/dL) at both 30 min (3.24 ± 0.52 μg/dL) and 60 min (3.27 ± 0.65 μg/dL) after induction. Compared with baseline, some of the T4 trends were inconsistent between animals, and individual variability in response was responsible for 86% of the overall variability. Regardless of the observed change under isoflurane anesthesia, all measurements in all prairie dogs and at all time points (2.4 to 4.4 μg/dL) were within the reported normal plasma T4 reference range for this species. In conclusion, isoflurane anesthesia appears to cause a significant but inconsistent reduction in plasma T4 concentrations in black-tailed prairie dogs, but because values remain within normal basal levels, the clinical importance of this effect is likely minimal.