Ovarian cancer is one of the most lethal gynecologic neoplasms. Even though various new chemotherapeutics have been developed for the treatment of ovarian cancer, drug resistance and undesired serious side effects remain unavoidable obstacles for chemotherapeutic approaches. New strategies to overcome the therapeutic dilemma are needed. Claudin-3 (CLDN3) is a recently discovered gene generally overexpressed in human ovarian cancers but not in normal ovarian tissue. Its high expression has been identified to associate with the invasion, proliferation and survival of cancer cells, making it a promising target for gene therapy of ovarian cancer. However, in gene therapy, traditional gene carriers such as virus or cationic liposomes suffer from distressing shortcomings of potential carcinogenicity, obvious cytotoxicity and immunogenicity. Nanoparticles (NPs) based on PLGA are a novel gene delivery system with good biodegradability, excellent biocompatibility and low toxcity for in vivo gene delivery compared with traditional gene carriers. We constructed a plasmid expressing shRNA targeted CLDN3 (pshCLDN3) encapsulated with PLGA-NPs, and administered it by i.p. injection to nude mice bearing intraperitoneal SKOV3 ovarian cancer, to investigate the antitumor potential of knocking down CLDN3. After 12 times of administration, the tumors of each group were compared. The underlying antitumor mechanisms were revealed by immunostaining of CD31, Ki-67 and TUNEL assay, to exhibit possible alterations in microvessel density, cell proliferation and cell apoptosis. Our study demonstrated that i.p. administration of pshCLDN3 effectively suppressed the expression of CLDN3 and, thus, inhibited the growth of ovarian tumors, significantly reducing tumor weight by 67.4% compared with blank controls (p<0.05). Immunostaining of CD31, Ki-67 and TUNEL assay demonstrated decreased angiogenesis (p<0.05), reduced proliferation (p<0.05) and increased apoptosis (p<0.05) in the pshCLDN3 treated group compared with controls. No obvious toxicity of PLGA-NPs was observed either in vitro or in vivo. Our results indicated that knockdown of CLDN3 by pshCLDN3 encapsulated in PLGA NPs may provide a promising approach for the treatment of ovarian cancer.