Plukenetia volubilis is a perennial woody oilseed vine, which belongs to Euphorbiaceae family. Seed oil of P. volubilis is rich in polyunsaturated fatty acids, especially the α-linolenic acid, which accounts for 44.2%-50.8%. Therefore, P. volubilis has the great potential to be an ideal feed stock for production of oil with a high nutritional value. However, P. volubilis still suffers from mortal diseases caused by Fusarium oxysporum, which considerably hinder the widely exploitation and utilization of products from P. volubilis. At present, it is difficult to breed new variety by traditional cross-breeding because of rare germplasm resources. Therefore, it is urgent to obtain new germplasm resources of P. volubilis. In this study, seeds of P. volubilis were irradiated with fast neutron (FN) for creating new germplasm. Firstly, the effects of different dose of fast neutron on seedling survival rates of the plants were investigated to determine the semi-lethal dose, which will be applied to construct the P. volubilis mutant library. The agronomic and other biological traits of the M1 plants were investigated. The results showed that: (1) The semi-lethal radiation dose of FN on P. volubilis seeds is approximately 47.6 Gy; (2) The M1 seedlings induced by fast-neutron radiation grew abnormally; (3) FN induces a range of abnormal phenotypes in P. volubilis, accounted for 9.2% of M1 surviving plants; (4) Five mutants were identified by random amplified polymorphic DNA (RAPD) analysis; (5) The fruit size of mutant 3A423 was significantly increased, and the average weight of its seed was 1.8-fold heavier than that of the wild-type (WT) plants. Thus, mutant 3A423 is an excellent candidate parent for breeding high-yielding P. volubilis. Taken together, these mutants obtained in this study will be useful for further research of genetic breeding and functional genomics of P. volubilis.