There is currently no causal treatment available for Parkinson's disease (PD). However, the use of glial cell line-derived neurotrophic factor (GDNF) to provide regenerative effects for neurons is promising. Such approaches require translational delivery systems that are functional in diseased tissue. To do so, we used a non-viral Sleeping Beauty (SB) transposon system to overexpress GDNF in adipose tissue-derived mesenchymal stromal cells (adMSCs). Entrapment of cells in fibrin hydrogel was used to boost potential neurorestorative effects. Functional GDNF-adMSCs were able to secrete 1066.8 ± 169.4 ng GDNF/120,000 cells in vitro. The GDNF-adMSCs were detectable for up to 1 month after transplantation in a mild 6-hydroxydopamine (6-OHDA) hemiparkinson male rat model. Entrapment of GDNF-adMSCs enabled GDNF secretion in surrounding tissue in a more concentrated manner, also tending to prolong GDNF secretion relatively. GDNF-adMSCs entrapped in hydrogel also led to positive immunomodulatory effects via an 83% reduction of regional IL-1β levels compared to the non-entrapped GDNF-adMSC group after 1 month. Furthermore, GDNF-adMSC-treated groups showed higher recovery of tyrosine hydroxylase (TH)-expressing cells, indicating a neuroprotective function, although this was not strong enough to show significant improvement in motor performance. Our findings establish a promising GDNF treatment system in a PD model. Entrapment of GDNF-adMSCs mediated positive immunomodulatory effects. Although the durability of the hydrogel needs to be extended to unlock its full potential for motor improvements, the neuroprotective effects of GDNF were evident and safe. Further motor behavioral tests and other disease models are necessary to evaluate this treatment option adequately.Keywords: Adipose tissue–derived mesenchymal stromal cells (adMSCs); Fibrin hydrogel; Neurodegeneration; Neurotrophic factors; Tissue engineering; Transplantation.