Various drugs are used to treat patients with neuropathic pain; however, optimal treatment using acetaminophen (A) and/or tramadol (T) remains unclear. The evidence supporting the drug choice and the timing of administration is insufficient. Therefore, the objective of the present study was to investigate the effect of T and/or A on pain-related behavior in a nucleus pulposus (NP) rat model. Sprague-Dawley rats (n = 180) were divided into NP-A (52mg/kg), NP-T (6mg/kg), NP-AT (combined A and T), NP-S (saline), and sham groups (n = 36 per group). The rats received 0.2mL of treatment solution orally once daily for 7days after application of NP on the left L5 dorsal root ganglion (DRG). Behavioral testing and immunohistochemistry analysis for some markers' expressions in DRGs and the spinal cord were performed. Pain thresholds in the NP-AT group did not significantly differ from the sham at all time points, while those were significantly lower in the NP-A and in the NP-T groups at D7 and/or D14 (p < 0.05). Tumor necrosis factor-α in the NP-S group was significantly higher at D2 and D7 (p < 0.05). Among the three treatment groups, activating transcriptional factor 3 and growth-associated protein 43 showed a tendency toward an increase at D7-D21. Combined administration of acetaminophen and tramadol maintained in the pain threshold in the rat NP model. These findings suggest that the combination of acetaminophen and tramadol might be a potential therapeutic modality for patients with lumbar disc herniation. These slides can be retrieved under Electronic Supplementary Material.