The aim of our study was to investigate the effects of a shortened daily photoperiod on anxiety-like behaviour, brain oxidative stress, lipid status and fatty acid composition of serum lipids in a streptozotocin (STZ)-induced model of diabetes mellitus in rats. Male Wistar rats were divided into the following groups: first group—control group (C12/12); second group—diabetic group (DM12/12; 100 mg/kg STZ); third group—control group exposed to a light/dark cycle 6/18 h (C6/18); fourth group—diabetic group exposed to a light/dark cycle 6/18 h (DM6/18). Anxiety-like behaviour was tested three weeks following STZ injection by elevated plus maze (EPM) and open-field test (OFT). Oxidative stress parameters were measured in the cortex, hippocampus and thalamus, while lipid status and fatty acid methyl esters (FAMEs) were measured in the serum. Both EPM and OFT showed a lower degree of anxiety-like behaviour in the DM6/18vs. DM12/12group. Lipid peroxidation in the cortex, hippocampus and thalamus was significantly lower in the DM6/18vs. DM12/12group (p< 0.05), associated with an increased level of antioxidant enzymes and protein thiols in the cortex and thalamus. In the DM6/18group, oleic, vaccenic, dihomo-γ-linolenic and docosahexaenoic acid concentrations were significantly higher in comparison to the DM12/12group. A shortened daily photoperiod alleviates anxiety-like behaviour in diabetic rats by reduced lipid peroxidation and changes in the serum fatty acids profile.