Abstract The purpose of this study was to investigate the analgesic interaction between matrine and paracetamol in an acetic acid-induced writhing model in mice. Fifty percent effective dose (ED50) values of the individual drugs were determined, and the different proportions of matrine and paracetamol were assayed using the isobolographic method. Our study demonstrated that both of matrine and paracetamol dose-dependently inhibited the writhing response evoked by acetic acid, and the ED50 values and their 95% confidence intervals against these tonic pain were 21.10 (17.86–24.92) mg/kg and 61.30 (50.71–74.10) mg/kg for matrine and paracetamol, respectively. At the fixed ratios of 1:1, 1:3 and 3:1, the experimental ED50 values of matrine and paracetamol combinations and their 95% confidence intervals were 10.52 (5.14–21.55) mg/kg, 9.13 (4.46–18.70) mg/kg and 4.98 (4.17–5.95) mg/kg, respectively, their theoretical ED50 values and 95% confidence intervals were 41.20 (36.31–46.74) mg/kg, 51.25 (44.19–59.44) mg/kg and 31.15 (27.25–35.60) mg/kg, and the experimental ED50 values of matrine and paracetamol combination were significantly lower than their calculated theoretical ED50 values (all P