Objective To investigate the autophagy inducing effect of dihydroartemisinin (DHA) on prostate cancer PC-3 cells and its possible mechanism. Methods PC-3 cells were treated with 0, 12.5, 25, 50, 100 μmol/L of DHA. Cell viability was detected by CCK-8 method, and cell proliferation rate was detected by cell clone formation assay. Set control group, DHA group (50 μmol/L DHA for 48 h), 3-MA group (5 mmol/L 3-MA for 48 h) and DHA+3-MA group (50 μmol/L DHA+5 mmol/L 3-MA for 48 h), expressions of autophagy-related protein [microtubule associated protein light chain 3B (LC3B), yeast Atg6 homologue (Beclin-1)] were detected by Western blotting and RT-qPCR, the cell viability was detected by CCK-8 method, and the apoptosis rate was detected by flow cytometry, the formation of autophagosomes was observed by transmission electron microscope. PC-3 cells were transfected with the autophagy double labeled lentivirus mCherry-GFP-LC3B to detect the changes of autophagy flow. Set control group, DHA group (50 μmol/L DHA for 48 h), NAC group (5 mmol/L NAC for 48 h) and DHA+NAC group (50 μmol/L DHA+5 mmol/L NAC for 48 h), expressions of ROS/AMPK/mTOR signaling pathway related proteins were detected by Western blotting. After treated with 50 μmol/L DHA for 48 h, the total protein was extracted and divided into Input group (whole protein lysate), IP group (added with Beclin-1 antibody), and IgG group (added with the same mass of IgG), interaction between Beclin-1, Vps34, Bcl-2 and HMGB1 in PC-3 cells was detected by the Co-IP. Results CCK-8 assay showed that the survival rate of PC-3 cells was decreased with the increase of the concentration of DHA in a dose- and time-dependent manner (P<0.05). The half inhibitory concentration (IC₅₀) of DHA for 24 h, 48 h and 72 h were 97.12, 57.10 and 29.35 μmol/L, select 50 μmol/L DHA for 48 h for follow-up experiments. Cell clone formation assay showed that the colony formation rate of PC-3 cells decreased significantly with the increase of DHA concentration (P<0.01). Western blotting and RT-qPCR results showed that, compared with control group, the mRNA and protein expression levels of Beclin-1, LC3B increased in PC-3 cells of DHA group (P<0.01); Compared with DHA group, the mRNA and protein expression levels of Beclin-1 and LC3B significantly decreased in PC-3 cells of DHA+3-MA group (P<0.01). Transmission electron microscopy showed that there were obvious autophagosomes in PC-3 cells of DHA group, and the number of autophagosomes was significantly increased compared with control group (P<0.05). The results of mCherry-GFP-LC3B lentivirus transfection showed that the ratio of red and yellow spots per cell in DHA group was higher (P<0.01) and that in DHA+3-mA group was lower (P<0.01). Compared with DHA group, the survival rate of DHA+3-mA group decreased (P<0.05) and the apoptosis rate increased (P<0.01). Compared with DHA group, the expression levels of p-mTOR decreased in PC-3 cells of DHA group (P<0.05), the expression levels of p-AMPK increased (P<0.01); Compared with DHA group, the expression levels of p-mTOR increased in PC-3 cells of DHA+NAC group (P<0.05), the expression levels of p-AMPK decreased (P<0.01). The results of Co-IP experiments showed that the effect of Beclin-1 on Bcl-2 was weakened and the binding with Vps34 and HMGB1 was enhanced after DHA treatment. Conclusions DHA can induce autophagy in prostate cancer PC-3 cells. The mechanism may be related to the regulation of the autophagy-related genes Beclin-1, LC3 and ROS/AMPK/mTOR signaling pathways.