Peripheral neuropathy represents an important complication of diabetes involving a spectrum of structural, functional and biochemical alterations in peripheral nerves. Recent observations obtained in our laboratory have shown that the levels of neuroactive steroids present in the sciatic nerve of rat raised diabetic by a single injection of streptozotocin (STZ) are reduced and that, in the same experimental model, treatment with neuroactive steroids, such as progesterone, testosterone and their derivatives show neuroprotective effects. On this basis, an interesting therapeutic strategy could be to increase the levels of neuroactive steroids directly in the nervous system. With this perspective, ligands of translocator protein-18 kDa (TSPO) may represent an interesting option. TSPO is mainly present in the mitochondrial outer membrane, where it promotes the translocation of cholesterol to the inner mitochondrial membrane, and, as demonstrated in other cellular systems, it allows the transformation of cholesterol into pregnenolone and the increase of steroid levels. In the diabetic model of STZ rat, we have here assessed whether treatment with Ro5-4864 (i.e., a ligand of TSPO) could increase the low levels of neuroactive steroids in sciatic nerve and consequently to be protective in this experimental model. Data obtained by liquid chromatography-tandem mass spectrometry show that treatment with Ro5-4864 was able to significantly stimulate the low levels of pregnenolone, progesterone and dihydrotestosterone observed in the sciatic nerves of diabetic rats. The treatment with Ro5-4864 also counteracted the impairment of NCV and thermal threshold, restored skin innervation density and P0 mRNA levels, and improved Na+,K+-ATPase activity. In conclusion, data here reported show for the first time that a TSPO ligand, such as Ro5-4864, is effective in reducing the severity of diabetic neuropathy through a local increase of neuroactive steroid levels.