Whilst the majority of familial medullary thyroid cancer (MTC) is caused by germline mutations of the RET proto-oncogene, there are families and individuals with predisposition to MTC in whom no RET mutation has been identified (non-RET MTC). Recently, we identified novel mutations in a single gene termed MTC2 in non-RET MTC individuals by whole exome sequencing. The precise role of these MTC2 germline mutations in MTC tumorigenesis is however unclear. Here, we examined the functional consequences of MTC2 mutants V128L and G318Afs*22 to determine their roles in MTC. Luciferase (LUC) reporter assays showed that MTC2-V128L retained transcriptional activity with a significant increase in LUC activity in response to steroid hormone receptor-agonist DPN in HCT116 (3.7-fold; P<0.01) and MCF-7 (1.8-fold; P<0.05) cells. In contrast, MTC2-G318Afs*22 was incapable of inducing LUC activity in either cell line (P=NS). Furthermore, MTC2-G318Afs*22 failed to inhibit ERα-driven luciferase activity in response to either 17β-estradiol (E2) or ERα-agonist PPT, or restrain ERα-driven proliferation of MCF7 cells (P=NS compared to ERα alone). In contrast, WT MTC2 and MTC2-V128L inhibited ERα-driven LUC activity (>60%; P<0.01) and cell proliferation (>30%; P<0.05). As RET expression is known to be stimulated by oestrogen, we then determined the influence of MTC2 mutants on RET in E2- and PPT-treated HCT116 cells. In contrast to WT MTC2, MTC2-G318Afs*22 was unable to oppose ERα-stimulation of the RET proto-oncogene at both the mRNA and protein level (P=NS compared to ERα alone). Treatment with anti-oestrogen 4-hydroxytamoxifen was however capable of inhibiting E2-induced RET mRNA expression in cells with MTC2-G318Afs*22. Together these data indicate an emerging role for MTC2 as a novel susceptibility gene in non-RET MTC development, especially as MTC2 mutant G318Afs*22 was associated with higher RET levels. These results also suggest that anti-oestrogens might represent a promising therapeutic strategy for MTC individuals with defective MTC2.