The second mammalian GnRH isoform (GnRH-II) and its specific receptor (GnRHR-II) are ubiquitously expressed, with elevated levels in the testis. Gene coding errors prevent their production in many species, but both genes are functional in swine. We demonstrated that GnRHR-II localizes to porcine Leydig cells, and exogenous GnRH-II robustly stimulates testosterone production in vivo, despite minimal luteinizing hormone (LH) secretion. These data suggest that GnRH-II directly effects steroidogenesis in the boar testis. To explore this hypothesis, we produced a GnRHR-II knockdown (KD) swine line. Upon characterisation of this line, serum testosterone concentrations were reduced in GnRHR-II KD compared with littermate control males during pubertal development. However, concentrations of LH were unaffected, indicating that GnRHR-II KD impairs steroidogenesis directly at the testis rather than inhibiting gonadotropin secretion from the anterior pituitary gland. Based on these results, the objective of this study was to compare diurnal secretory patterns of testosterone in mature GnRHR-II KD (n = 5) and littermate control (n = 5) males. Boars were fit with indwelling jugular cannulae and blood was collected every 15 min for 8 h. Serum was assayed for testosterone concentration via radioimmunoassay. Next, GnRHR-II KD (n = 5) and littermate control (n = 4) boars were killed, testis weight was recorded, and testicular tissue was collected for RNA isolation. To confirm KD in these animals, digital droplet PCR was performed to quantify GnRHR-II mRNA abundance (normalized to β-actin). Data were analysed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) with line (transgenic or control) as the fixed effect and litter as a random effect. For hormone data, time and line × time interaction were included as fixed effects, with time as a repeated measure. Although there was no effect of time or line × time interaction (P > 0.05) on serum testosterone concentrations, we observed a line effect (P < 0.05). Differences between lines were dramatic; testosterone was reduced by 82% in GnRHR-II KD (0.75 ± 0.05 ng/mL) compared with littermate control (4.09 ± 0.29 ng/mL; P < 0.05) males. Despite divergent testosterone levels, testis weights were similar between lines (P > 0.05) indicative of altered Leydig cell function as opposed to hypertrophy/hyperplasia. Given that testicular GnRHR-II mRNA levels were reduced by 69% in transgenic animals (P < 0.001), these data demonstrate that GnRH-II and its receptor play a critical role in testosterone biosynthesis within porcine Leydig cells. Thus, this report reveals novel mediators of testicular function in the boar and challenges the central dogma of testosterone regulation. Because testosterone dictates male reproductive success, GnRH-II and its receptor represent unique targets to improve fertility in swine.