In the present study, we investigated the association of prolidase activity in streptozotocin (STZ) induced diabetic rat model with hippoctunpus-dependent spatial memory functions in chronic hyperglycemic conditions. Towards this, a single dose of STZ (30 mg/kg, i.p.) was administered to 4-month-old female Sprague Dawley rats and hyperglycemia was confirmed in all the STZ treated rats. To assess the spatial memory, at the time intervals of 9 days, 30 days and 90 days post STZ, administration, the animals were subjected to Barnes maze task. Prolidase activity and glucose concentration in the serum were measured at these time points. With prolonged duration of hyperglycemia, severe impairment in spatial memory functions was seen in STZ induced diabetic rats which was correlatable with reduction in prolidase levels (P <0.05), thus suggesting that alterations in synaptic integrity due to prolidase mediated extracellular matrix remodeling as the possible mechanism of memory defects observed during chronic diabetes.