Morphine (MOR) is a commonly prescribed drug for the treatment of moderate to severe diabetic neuropathic pain (DNP). However, long-term MOR treatment is limited by morphine analgesic tolerance (MAT). The activation of microglial cells and the release of glia-derived proinflammatory cytokines are known to play an important role in the development of MAT. In this study, we aimed to investigate the effects of the dipeptidyl peptidase-4 inhibitor (DPP-4i) teneligliptin (TEN) on MOR-induced microglial cell activation and MAT in DNP rats. DNP was induced in four groups of male Wistar rats through a single intraperitoneal injection of streptozotocin (STZ) (50 mg/kg, freshly dissolved in 5 mmol/L citrate buffer, pH 4.5). Sham rats were administered with the vehicle. Seven days after STZ injection, all rats were implanted with an intrathecal (i.t) catheter connected to a mini-osmotic pump, divided into five groups, and infused with the following combinations: sham + saline (1 µL/h, i.t), DNP + saline (1 µL/h, i.t), DNP + MOR (15 µg/h, i.t), DNP + TEN (2 µg/h, i.t), and DNP + MOR (15 µg/h, i.t) + TEN (2 µg/h, i.t) for 7 days at a rate of 1 μL/h. The MAT was confirmed through the measurement of mechanical paw withdrawal threshold and tail-flick tests. The mRNA expression of neuroprotective proteins nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase-1 (HO-1) in the dorsal horn was evaluated by quantitative PCR (qPCR). Microglial cell activation and mononucleate cell infiltration in the spinal cord dorsal horn were assessed by immunofluorescence assay (IFA) and Western blotting (WB). The results showed that co-infusion of TEN with MOR significantly attenuated MAT in DNP rats through the restoration of neuroprotective proteins Nrf2 and HO-1 and suppression of microglial cell activation in the dorsal horn. Though TEN at a dose of 2 μg has mild antinociceptive effects, it is highly effective in limiting MAT.