Castration is a routine procedure performed on beef and dairy operations in the U.S. All methods of castration cause behavioral, physiologic, and neuroendocrine changes associated with pain. The American Veterinary Medical Association and the American Association of Bovine Practitioners recommend that anesthesia and analgesia be administered at castration. The objective of this study was to evaluate the effectiveness of bupivacaine liposome suspension a novel, long-acting, local anesthetic administered as a nerve block at castration. The authors chose to investigate this novel formulation as an alternative to current industry standards using lidocaine nerve blocks alone, or in combination with meloxicam. Thirty male Holstein calves, 16-20 weeks of age, were enrolled and randomly assigned to 1 of 4 treatment groups prior to surgical castration: 1) bupivacaine liposome suspension block + oral placebo (BUP); 2) lidocaine block + oral placebo (LID); 3) lidocaine block + oral meloxicam (1 mg/kg) (LID + MEL); and 4) saline block + oral placebo (CON). Biomarkers were collected at -24 h and from 0 to 120 hours post-castration and included infrared thermography, pressure mat gait analysis, chute defense and behavior scoring (pain and activity), and blood sampling for serum cortisol and prostaglandin E2 metabolites (PGEM). Responses were analyzed using repeated measures, with calf nested in treatment as a random effect, and treatment, time, and their interaction designated as fixed effects. Results from pressure mat gait analysis show the CON had a shorter front limb stance time from baseline (-8.73%; 95% CI: -24.84 to 7.37%) compared to BUP and LID + MEL (> 5.70%; 95% CI: -22.91 to 23.79%) (P < 0.03). The CON tended to have an increase in front limb force from baseline (6.31%; 95% CI: -1.79 to 14.41%) compared to BUP, LID, and LID + MEL (< -5.06%; 95% CI: -14.22 to 0.95%) (P < 0.04). The CON displayed higher counts of hunched standing (2.00; 95% CI: 1.68 to 2.32) compared to LID + MEL (1.43; 95% CI: 1.13 to 1.72) (P = 0.05). The CON had higher cortisol concentrations at 24 h (7.70 ng/mL; 95% CI: 1.52 to 13.87 ng/mL) relative to BUP (3.11 ng/mL; 95% CI: -2.56 to 8.79 ng/mL) (P = 0.002). At 4 and 24 h, LID + MEL had lower PGEM concentrations from baseline (-32.42% and -47.84%; 95% CI: -78.45 to -1.80%) compared with CON (27.86% and 47.63%; 95% CI: 7.49 to 82.98%) (P < 0.02). Administration of bupivacaine liposome suspension as a local anesthetic block at the time of castration was as effective at controlling pain as a multi-modal approach of lidocaine and meloxicam.