Abstract To investigate the enteric neuropathy in diabetic rats and the role of glia cell line-derived neurotrophic factor (GDNF) and its signalling pathway PI3K/Akt in regulating enteric neurons survival. Male Sprague–Dawley (SD) rats were randomly divided into normal control group, diabetic groups (rats with diabetes for 4, 8 and 12 weeks respectively). Proximal and distal colon specimens were obtained from each rat. Phosphoinositol-3-kinase signalling pathway was analysed by Akt phosphorylation. Protein gene product 9.5 (PGP9.5) used as a pan-neuronal marker. The expressions of GDNF, phospho-Akt (p-Akt), neuronal nitric oxide synthase (nNOS) neurons, cholinergic [choline acetyltransferase (CHAT) stained] neurons and total neurons were measured by immunohistochemical streptavidin-biotin complex (SABC) methods, Western blot and real-time polymerase chain reaction methods for each specimen. (i) Expression of GDNF was significantly decreased in diabetes 8 and 12 weeks group compared with the control group in both proximal (P < 0.01) and distal (P < 0.01) colon. The change of GDNF expression was greater in the 12 weeks group than that in the 8 weeks group (P < 0.05). There were no significant differences between the 4 weeks group and the control group in expression of GDNF (P > 0.05). (ii) The change trend of Akt phosphorylation was the same with GDNF. (iii) The numbers of nNOS, CHAT neurons and total neurons in proximal and distal colon were decreased significantly during the course of diabetes (P < 0.05). Diabetes can significantly induce enteric neuropathy. This change may be mediated, in partly, via a reduction of GDNF and its main downstream signalling pathway PI3K/Akt, which is a survival signal for enteric neurons.