The present study to attempt to cultivate Angiostrongylus cantonensis from third-stage larvae (AcL3) to fourth-stage larvae (AcL4) in vitro in defined complete culture medium that contained with Minimum Essential Medium Eagle (MEM), supplemented amino acid (AA), amine (AM), fatty acid (FA), carbohydrate (CA) and 20% fetal calf serum (FCS) was successful. When AcL3 were cultured in the defined complete culture medium at 37°C in a 5% CO2 atmosphere, the larvae began to develop to AcL4 after 30 days of cultivation, and were enclosed within the sheaths of the third molts of the life cycle. Under these conditions, the larvae developed uniformly and reached to the fourth-stage 36 days. The morphology of AcL3 develop to AcL4 were recording and analyzing. Then comparison of A. cantonensis larval morphology and development between in vitro cultivation in defined complete culture medium and in vivo cultivation in infective BALB/c mice. The larvae that had been cultivated in vitro were smaller than AcL4 of infective BALB/c mice. However the AcL3 that were cultured using defined incomplete culture medium (MEM plus 20% FCS with AA+AM, FA, CA, AA+AM+FA, FA+CA, CA+AA+AM or not) did not adequately survive and develop. Accordingly, the inference is made that only the defined complete medium enable AcL3 develop to AcL4 in vitro. Some nematodes have been successfully cultured into mature worms but only a few researches have been made to cultivate A. cantonensis in vitro. The present study is the first to have succeeded in developing AcL3 to AcL4 by in vitro cultivation. Finally, the results of in vitro cultivation studies herein contribute to improving media for the effective development and growth of A. cantonensis. The gap in the A. cantonensis life cycle when the larvae are cultivated in vitro from third-stage larvae to fourth-stage larvae can thus be solved.