Autophagy is an important element in tumor progression. The clinical and commercial success of monoclonal antibodies (MAbs) for cancer has emerged as the fastest growing therapy. Prostate cancer is well appropriately suited for antibody therapy. PC322 is our newly prepared prostate cancer monoclonal antibody. In our current study, we described the impacts of PC322 on autophagy and biological behaviors in vitro and explored the possible mechanism of PC322 in prostate cancer cell autophagy. Role of PC322 on autophagy of LNCaP and C4-2 cells was observed with fluorescence microscopy, western blotting, and flow cytometry. Further, the function of PC322 on biological behaviors of prostate cancer cells was detected by CCK-8 kit, Hoechst 33342, wound healing assay and cell adhesion assay.... Finally, we found that Autophagy-related genes3 (ATG3) was up-regulated with stimulation of Rapamycin (RAPA), while PC322 inhibited ATG3 expression. In summary, in vitro experiments demonstrated that the new monoclonal antibody PC322 inhibit cell autophagy, proliferation, and metastasis of LNCaP and C4-2 cells, and promote their apoptosis. PC322 reduces ATG3 expression. Thereby suppresses the formation of autophagosomes by inhibiting LC3-I converting to LC3-II.