ObjectiveTo evaluate a regional anesthetic technique for blocking the abdominal midline in horses. Study designAnatomical description and prospective, crossover, placebo-controlled, blinded study. AnimalsAdult horses; two cadavers, six healthy animals. MethodsIn stage 1, 0.5% methylene blue with 0.25% bupivacaine (0.5 mL kg–1) was injected using ultrasonography into the internal rectus abdominis sheath (RAS) of two cadavers with a one-point or two-point technique. The dye spread was described after the dissection of the abdomens. In stage 2, each horse was injected with 1 mL kg–1 of 0.9% NaCl (treatment PT) or 0.2% bupivacaine (treatment BT) using a two-point technique. The abdominal midline mechanical nociceptive threshold (MNT) was measured with a 1 mm blunted probe tip and results analyzed with mixed-effect anova. Signs of pelvic limb weakness were recorded. ResultsThe cadaver dissections showed staining of the ventral branches from the eleventh thoracic (T11) to the second lumbar (L2) nerve with the one-point technique and T9–L2 with the two-point technique. Baseline MNTs were, mean ± standard deviation, 12.6 ± 1.6 N and 12.4 ± 2.4 N in treatments PT and BT, respectively. MNT increased to 18.9 ± 5.8 N (p = 0.010) at 30 minutes, and MNT was between 9.4 ± 2.0 and 15.3 ± 3.4 N from 1 to 8 hours (p > 0.521) in treatment PT. MNTs in treatment BT were 21.1 ± 5.9 to 25.0 ± 0.1 N from 30 minutes to 8 hours (p < 0.001). MNTs after the RAS injections were higher in treatment BT than PT (p = 0.007). No pelvic limb weakness was observed. Conclusions and clinical relevanceAntinociception of at least 8 hours without pelvic limb weakness was observed in the abdominal midline in standing horses after the RAS block. Further investigations are necessary to evaluate suitability for ventral celiotomies.