Objective To investigate the influences of hypericin (Hyp) on renal autophagy and AMPK/mTOR/ULK1 pathway in nephrotic syndrome (NS) rats. Methods Thirty-two 6-week-old SD rats were grouped into normal group (N group), NS group, Hyp group (60 mg/kg Hyp), Hyp AMPK inhibitor group (Hyp CC group) (60 mg/kg Hyp group) 0.2 mg/kg CC), 8 per group. After administration, an automatic analyzer was applied to detect the levels of 24-h urine total protein (UTP), blood urea nitrogen (BUN), serum creatinine (Scr) and albumin (ALB); HE staining and TEM observation were applied to observe renal pathological morphology and ultrastructure; Western blot was applied to detect the expression of autophagy, podocyte and AMPK/mTOR/ULK1 pathway proteins in kidney; immunofluorescence staining was applied to visualize the localization of autophagosomes and podocytes. Results Compared with the N group, the glomerular volume of the NS group increased, the renal tubules atrophied or partially disappeared, and the basement membrane thickened; UTP, BUN, Scr, basement membrane thickness, foot process width and the ratio of p-AMPK/AMPK were obviously increased (P<0.05), the protein levels of ALB, LC3-II/I, Beclin-1, Atg5, Atg7, NPHS2, the relative fluorescence intensity of NPHS2 and Beclin-1, and the ratios of p-AMPK/AMPK and p-ULK1/ULK1 were obviously decreased (P<0.05). Compared with NS group, Hyp treatment was able to improve glomerular morphology, decrease UTP, BUN, Scr, basement membrane thickness, foot process width and ratio of p-AMPK/AMPK (P<0.05), increase protein levels of ALB, LC3-II/I, Beclin-1, Atg5, Atg7, NPHS2, the relative fluorescence intensity of NPHS2 and Beclin-1 and the ratios of p-AMPK/AMPK and p-ULK1/ULK1 (P<0.05). AMPK inhibitor group CC could attenuate the autophagy-promoting and kidney-protecting effects of Hyp. Conclusion Hyp may enhance the autophagy activity of renal cells and attenuate renal pathology such as podocyte injury in NS rats by activating the AMPK/mTOR/ULK1 pathway.