--- - |2+ Abstract Aim Matrix metalloproteinase‐9 (MMP9) plays a profibrotic role in renal fibrosis. Neutrophils produce MMP9 in many pathologic models. However, the effect of neutrophil on the progression of renal fibrosis and the relationship of MMP9 to the infiltration of neutrophils into the kidney remain unknown. Methods The surgery of unilateral ureter obstruction (UUO) was performed in male C57BL/6 mice. Kidneys were collected for analyses on days 0, 1, 3, 5 or 7 following surgery. The inflammatory cells were analysed by flow cytometry. The mRNA and protein levels of renal fibrosis factor and inflammatory factor were measured by qRT‐PCR, immumofluorescence and western blot analysis. Results In a mouse kidney model of UUO, neutrophil infiltration significantly increased and neutrophil accumulation reached the highest level at 5 days after the injury. In the obstructed kidney, depleting neutrophils decreased the expression of inflammatory factors, inhibited the accumulation of macrophages including type 2 macrophages and suppressed renal fibrosis. Almost all neutrophils produced MMP9 at the early stage of kidney obstruction. MMP9 attracted neutrophils and inflammatory cells because inhibiting MMP9 suppressed the infiltration of neutrophils and other inflammatory cells and reduced renal fibrosis, regardless of using MMP9 neutralizing antibody or MMP9 inhibitor or different intervening periods of days (0‐6, 0‐3 or 3‐6 were applied after kidney obstruction). Conclusion MMP9 promotes neutrophil infiltration by increasing the inflammatory level, macrophage accumulation and renal fibrosis in the obstructed kidney. Inhibiting MMP9 or depleting neutrophils in the early stage of acute kidney injury can relieve the progression of kidney fibrosis. - 'Acta Physiologica, Volume 227, Issue 2, October 2019. '