Parkinson’s disease (PD) is characterised by the preferential loss of dopaminergic neurones from the substantia nigra (SN) that leads to the hallmark motor disturbances. Animal and human studies suggest a beneficial effect of oestrogen to the nigrostriatal system, and the regulation of neurotrophic factor expression by oestrogens has been suggested as a possible mechanism contributing to that neuroprotective effect. The present study was designed to investigate whether the neuroprotection exerted by 17β-oestradiol on nigrostriatal dopaminergic neurones is mediated through the regulation of glial cell line-derived neurotrophic factor (GDNF) expression. Using an in vivo rat model of PD, we were able to confirm the relevance of 17β-oestradiol in defending dopaminergic neurones against 6-hydroxydopamine (6-OHDA) toxicity. 17β-oestradiol, released by micro-osmotic pumps, implanted 10 days before intrastriatal 6-OHDA injection, prevented the loss of dopaminergic neurones induced by 6-OHDA. 17β-oestradiol treatment also promoted an increase in GDNF protein levels both in the SN and striatum. To explore the relevance of GDNF increases to 17β-oestradiol neuroprotection, we analysed, in SN neurone-glia cultures, the effect of GDNF antibody neutralisation and RNA interference-mediated GDNF knockdown. The results showed that both GDNF neutralisation and GDNF silencing abolished the dopaminergic protection provided by 17β-oestradiol against 6-OHDA toxicity. Taken together, these results strongly identify GDNF as an important player in 17β-oestradiol-mediated dopaminergic neuroprotection.