Objective  To study the effect of reactive oxygen species (ROS)/p38 MAPK cascade reaction on the formation of kidney stones (KS) in rats and explore the mechanism.  Methods   Fifty SD rats were randomly divided into control group (normal feeding), N-acetylcysteine (NAC) group (intraperitoneally injected 200 mg/kg NAC), KS group (constructed calcium oxalate KS model), KS+NAC group (constructed calcium oxalate KS model, intraperitoneally injected 200 mg/kg NAC), KS+NAC+tunicamycin (TM) group(constructed calcium oxalate KS model, intraperitoneally injected 200 mg/kg NAC and 1 mg/kg TM), with 10 rats in each group. After 4 weeks of administration, 24 hours urine volume and oxalic acid (Ox) of each group were measured, serum creatinine (Cr), urea nitrogen (BUN) and uric acid (UA) levels were detected by automatic biochemical analyzer. HE staining and Von Kossa staining were used to observe the histopathological changes and crystal deposition of the kidney. TUNEL staining was used to detect apoptosis of renal tissue cells. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in renal tissue were measured by the kit. DHE fluorescent probes detected the levels of reactive oxygen species (ROS) in kidney tissue. Immunohistochemical staining was used to detect the expressions of microtubule-associated protein 1 light chain 3 B (LC3B) and glucose regulatory protein 78 (GRP78) in renal tissue, and the protein expression of LC3Ⅱ/LC3Ⅰ, Beclin1, GRP78, CCAAT/enhancer binding protein homologous protein (CHOP) in renal tissue was determined by Western blotting.  Results   Compared with the KS group, Ox in KS+NAC group decreased (P<0.05), BUN, Cr and UA levels decreased (P<0.05), renal tubule dilatation and calcium oxalate crystallization decreased, TUNEL positive cell rate decreased (P<0.05), SOD activity increased and MDA content decreased (P<0.05), ROS levels decreased (P<0.05), LC3B and GRP78 positive staining levels decreased (P<0.05), the relative protein expressions of p-p38 MAPK/p38 MAPK, LC3Ⅱ/LC3Ⅰ, Beclin1, GRP78 and CHOP were down-regulated(P<0.05). Compared with the KS+NAC group, Ox in KS+NAC+TM group increased (P<0.05), BUN, Cr and UA levels also increased (P<0.05), renal tubule dilated significantly, calcium oxalate crystals increased, TUNEL positive cell rate increased (P<0.05), SOD activity decreased and MDA content increased (P<0.05), ROS levels increased (P<0.05), LC3B and GRP78 positive staining levels increased (P<0.05), the relative protein expressions of p-p38 MAPK/p38 MAPK, LC3Ⅱ/LC3Ⅰ, Beclin1, GRP78 and CHOP were also up-regulated (P<0.05).  Conclusion   ROS/p38 MAPK cascade is involved in promoting KS formation in rats, which is related to the activation of endoplasmic reticulum stress-mediated autophagy pathway.