ABSTRACT Background Study aimed to obtain insights into physiological responses to immunocastration in pubertal boars by evaluating effects of alternative vaccination protocols and identifying a reliable immunocastration biomarker. Objectives It was hypothesized that the timing of gonadotropin‐releasing hormone (GnRH) suppression by immunocastration differentially affects reproductive function, as reflected by testicular histology and expression of selected genes related to testicular function, steroid metabolism, and Leydig cell differentiation and function. Materials and Methods Effects of three vaccination protocols on antibody titers, testicular histomorphology, and mRNA expression in immunocastrates slaughtered 4, 8, or 12 weeks (IC‐4, IC‐8, and IC‐12, respectively; n = 6 per group) after booster were compared with entire males (EMs; n = 6). Principal component analysis and hierarchical clustering were used to evaluate individual responses and to identify an immunocastration biomarker(s). The selected biomarker was also validated on samples from previous studies. Results All immunocastrated boars reacted immunologically to vaccination (increased GnRH antibody titer; p < 0.004). There was an increased nucleus‐to‐cytoplasm ratio in Leydig cells ( p < 0.033) and decreased testosterone concentration ( p < 0.041) in IC‐4 compared with EM, whereas values in IC‐8 and IC‐12 were intermediate. Hierarchical clustering differentiated immunocastrates with low testosterone (IC‐LT; median 0.56 ng/mL) and high testosterone (IC‐HT; median 7.04 ng/mL). In IC‐LT, expression levels of FSHR and ESR2 were higher than in IC‐HT, and those of LHCGH, STAR and INSL3 were lower compared with IC‐HT and EM, whereas expression of ESR1 and HSD17β7 was lower in IC‐LT and IC‐HT compared with EM (fold change > 1.5 and p < 0.05). Conclusion There were variable responses to immunocastration in pubertal boars subjected to three vaccination protocols. Suppression of testicular function was most pronounced in boars slaughtered 4 weeks after the booster, whereas progressive recovery occurred in some boars 8 and 12 weeks after the booster. Irrespective of vaccination protocol, INSL3 mRNA expression was a reliable immunocastration biomarker.