Mice are commonly anesthetized (or sedated) with drugs injected via the intraperitoneal or subcutaneous interscapular routes. The Governing Vessel 20 (GV20) site, located at the top of the head, is an alternative subcutaneous injection route, shown to be successful in dogs and cats. In a multicenter, randomized, blinded study design, C57BL/6 mice (n = 66: 60 males, 6 females) were assigned to injection with ketamine (60 mg/kg) and xylazine (5 mg/kg). Injection distributions were as follows: site 1, GV20 (n = 10) and intraperitoneal (n = 9); site 2, GV20 (n = 16), interscapular subcutaneous (n = 15), and intraperitoneal (n = 16). Outcome measures were times to ataxia, sternal recumbency, and the loss of righting reflex. Data from sites 1 and 2 were pooled following confirmation that there were no significant differences (Mann–Whitney test). Outcome measures were compared between injection routes with a Kruskal–Wallis test followed by a Dunn multiple comparisons test. Results are reported as median (range) times. Intraperitoneal injection was faster acting than the other injection routes, and there were no significant differences between the GV20 and interscapular subcutaneous routes: ataxia (GV20, 187.0 s [120 to 272]; subcutaneous, 165.0 s [120 to 372]; intraperitoneal, 88.5 s [58 to 171]), sternal recumbency (GV20, 305.5 s [153 to 400], subcutaneous, 305.0 s [249 to 384]; intraperitoneal, 184.5 s [120 to 397]), loss of righting reflex (GV20, 399.0 s [208 to 589]; subcutaneous, 347.0 s [249 to 468]; intraperitoneal, 190.0 s [142 to 402]). In summary, the GV20 subcutaneous injection route does not appear to have benefits compared with the intraperitoneal route and is not superior to the interscapular subcutaneous injection route in C57BL/6 mice when evaluated using a combination of ketamine and xylazine.