The present study aimed to investigate the potential role of microRNA-141-3p (miR-141-3p) in chronic inflammatory pain (CIP) by targeting the high-mobility group box1 (HMGB1) gene. In the in vitro study, BV2 microglial cells were selected and assigned into blank, lipopolysaccharide (LPS), miR-141-3p mimics, mimics control, miR-141-3p inhibitor, inhibitor control, miR-141-3p mimics+LPS, mimics control+ LPS, miR-141-3p inhibitor+LPS and inhibitor control+LPS groups. Ninety-six rats were randomly divided into 8 groups (12 rats in each group): blank control, model control, negative control (NC), miR-141-3p mimics+ complete Freund's adjuvant (CFA), mimics control+CFA, HMGB1 short hairpin RNA (shRNA)+CFA, HMGB1 NC+CFA and miR-141-3p mimics+HMGB1 shRNA+CFA groups. The quantitative real-time PCR, western blotting, enzyme-linked immunosorbent assay and pain behavioral test were used to measure the miR-141-3p and HMGB1 mRNA expressions, HMGB1 protein expression, inflammatory cytokines levels, and thermal and mechanical pain thresholds, respectively. Compared with the blank, mimics control, inhibitor control and miR-141-3p mimics+LPS groups, the miR-141-3p mimics group had increased miR-141-3p expression and interleukin (IL)-10 levels, and had decreased mRNA and protein expressions of HMGB1 and the levels of IL-1β, tumor necrosis factor-α (TNF-α) and IL-6, whereas the opposite trend were found in the LPS, miR-141-3p inhibitor, mimics control+LPS and inhibitor control+LPS groups. Compared with the LPS, miR-141-3p inhibitor, mimics control+LPS and inhibitor control+LPS groups, the miR141-3p+LPS group had an obviously decreased expression of miR-141-3p and IL-10, increased mRNA and protein expressions of HMGB1 and the levels of IL-1β, TNF-α and IL-6. Compared with the rats in the blank control group, the miR-141-3p expression, IL-10 level, and thermal and mechanical pain thresholds decreased significantly, whereas the mRNA and protein expressions of HMGB1, IL-1β, TNF-α and IL-6 increased significantly in rats in the NC, mimics control+CFA and HMGB1 NC+ CFA groups. The miR-141-3p expression was increased in rats in the miR-141-3p mimics+HMGB1 shRNA+CFA group. Our study demonstrated that miR-141-3p can alleviate the CIP by downregulating the downstream target gene HMGB1.