Objective To investigate the role and mechanism of miR-216a-5p in high mobility group protein-B1 (HMGB1)-mediated peritoneal dialysis-associated peritonitis (PDAP) induced by Staphylococcus epidermidis infection in mice. Methods Healthy male C57BL/6J mice were selected and randomly divided into control group, infection group, and infection+HMGB1 inhibitor group, peritoneal effusion and peritoneal tissue was collected for detection of whit blood cell (WBC) count, HE and immunohistochemical staining; mRNA and protein expression levels of interleukin-1α(IL-1α), interleukin-6 (IL-6), tumor necrosis factors-α(TNF-α), HMGB1, NF-κB, and I-κBi were detected by real-time polymerase chain reaction (RT-PCR) and Western Blot; bioinformatics prediction found that miR-216a-5p may bind with HMGB1 and participate in the occurrence of HMGB1-mediated PDAP, and construct dinfection+miR-216a-5p mimics mice group, relationship between miR-216a-5p and HMGB1 was verified by double luciferase reporter gene assay, changes in expression of HMGB1 were detected by RT-PCR, Western blot and immunohistochemical staining. Results Compared with control group, WBC count of peritoneal effusion in mice in infection group increased, inflammatory infiltration was obvious, expression of mRNA and protein of IL-1α, IL-6, TNF-α and HMGB1 all increased (all P < 0.05); after intervention by HMGB1 inhibitor (glycyrrhizin), WBC count of mice peritoneal effusion decreased, inflammatory infiltration improved, expression of mRNA and protein of IL-1α, IL-6, TNF-α and HMGB1 all decreased (all P < 0.05). The expression of NF-κB in mice in infection group was higher than that in control group, I-κBi was lower than that in control group; after HMGB1 inhibitor intervention, NF-κB expression decreased, I-κB expression increased (both P < 0.05). RT-PCR results confirmed that the content of miR-216a-5p decreased significantly in infection group compared with control group; dual-luciferase reporter gene assay suggested that miR-216a-5p could directly act on the 3' UTR region of HMGB1; compared with infection group, the expression of mRNA and protein of HMGB1 decreased in mice in infection+miR-216a-5p mimics group (both P < 0.05). Conclusion HMGB1 plays an important role in PDAP induced by Staphylococcus epidermidis infection, inhibiting HMGB1 can improve the inflammatory response in mice, miR-216a-5p can participate in the occurrence of PDAP induced by Staphylococcus epidermidis infection through targeting HMGB1.