Angus crossbred bulls (n = 60; 257 ± 5.4 d of age; initial BW 358.8 ± 3.78 kg) were used to study the effect of a vaccine against gonadotropin-releasing factor (GnRF) and band castration on behavioral and physiological indicators of pain. Cattle were randomly assigned to 1 of 3 treatments: bulls, band-castrated calves without pain mitigation (castrated), and immune-vaccinated animals administered an anti-GnRF vaccine (vaccinated). All animals were fitted with a radio frequency ear tag so that individual animal feed intake and feeding behavior were recorded daily over the entire trial using an electronic feed bunk monitoring system. Two doses of anti-GnRF vaccine were administrated on d -35 and 0 and band castration was performed on d 0. Animal BW was recorded weekly starting on d -36 until d 56. Visual analog scores (VAS) were measured on d -36 -35, -1, and 0, and salivary cortisol concentration was measured at -30, 0, 30, 60, 120, and 270 min on d -35 and 0 after castration. Saliva and blood were obtained on d 1, 2, 5, and 7 and weekly until d 56 for determination of cortisol and complete blood cell count. Video data were collected for pain, sexual, and aggressive behavior daily the first week and once a week until d 56. Data were analyzed with a mixed-effect model with castration, time, and their interactions as main effects. Vaccinated calves had reduced ADG and intake (P < 0.05 and P < 0.001, respectively) during the first week after vaccination. Band-castrated calves had reduced ADG and intake (P < 0.001) until the end of the study. No differences in salivary cortisol and VAS were observed among groups at d -35 after the first vaccination and before band castration. However, on d 0, castrated cattle had greater cortisol concentrations and VAS (P < 0.05 and P < 0.01, respectively) than bulls and vaccinated animals. Complete blood cell count did not differ (P > 0.05) between treatments on d 0, 1, and 2. At d 56, vaccinated calves had greater (P < 0.05) final BW than band-castrated calves and both had less final BW than bulls. There was no indication that vaccination caused any physiological or behavioral changes indicative of pain. In contrast, band castration resulted in elevated cortisol scores and VAS indicative of a pain response and behavior related to pain (P < 0.001) until d 42 of the study. The present study demonstrates that anti-GnRF vaccine is a viable animal welfare-friendly alternative to traditional band castration in beef cattle under North American feedlot practices.