This study evaluated the effects of the methanolic extract of Guibourtia tessmannii (GT) and selenium (Se) on cell viability, intracellular calcium concentration ([Ca2+](i)), apoptosis and oxidative stress through transient receptor potential vanilloid 1 (TRPV1) channel activity in CCL-97 (R2C) tumour Leydig cells. The cells were divided into nine groups and treated as follows: (a)-Control, (b)-Capsazepine (CPZ, 0.1 mM, a TRPV1 channel blocker), (c)-Capsaicin (CAP, 0.01 mM, a TRPV1 channel activator), (d)-GT (500 mu g/ml), (e)-GT+CPZ, (f)-GT+CAP, (g)-Se (200 nM), (h)-Se+CPZ and (i)-Se+CAP. After treatments, cell viability, [Ca2+](i), apoptosis, caspase 3/9, reactive oxygen species (ROS) and mitochondrial membrane depolarisation (MMD) were evaluated. The [Ca2+](i), apoptosis, caspase 3/9, MMD and ROS levels were significantly (p < 0.001) increased in CAP group, but lowered in CPZ group. Interestingly, these parameters were significantly (p < 0.001) improved by GT and Se, compared to the CAP group. Moreover, the co-administration of GT+CAP or Se+CAP inhibited the cytotoxicity of CAP. Thus, the modulatory properties of GT and Se on Ca2+ influx, apoptosis and oxidative stress require the integrity of TRPV1 channel in CCL-97 Leydig cells. These results suggest that GT and Se might be used in the management of cytotoxicity in the testes, involving TRPV1 channel activity.