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[Objectives] To determine the optimal reproduction and storage conditions for the Hyphantria cunea nudeopolyhedrovirus (HcNPV) in order to provide a theoretical basis and technological support for the commercial production of this virus. [Methods] The effects of different larval instars, virus concentration, duration, temperature and population density, were analyzed to identify the optimal condition for HcNPV reproduction in vivo. Effects of different formulations, times, temperatures and adjuvants on the stability of HcNPV storage were also investigated to determine the optimal HcNPV bioactivity assay method. In feeding bioassays, the number of proliferating occlusion bodies (OB) were recorded with a hemocytometer, and the lethal time (LT50) was calculated to quantify the insecticidal activity of HcNPV Third instar larvae fed a diet with a HcNPV concentration of 1x106 OBmL-1 were used for follow-up experiments. [Results] OB proliferation in 5th instar larvae reared on an artificial diet containing HcNPV at a concentration of 1x106 OBmL-1 was significantly higher (1.16x1010 OB per larva) than in other treatments. The optimal temperature for HcNPV reproduction was 20 °C, which resulted in 5.06x109 OB per larva. The abundance of HcNPV virions significantly decreased from 3.42x109 OB per larva after feeding for 1 day to 0.97x109 OB per larva after feeding for 5 days. Larvae kept at a density of 100 larvae/box (0.05 larvae/cm²) had 4.94x109 OB per larva, which was significantly higher than that of those kept at densities of 200, 300, 400, 500 larvae/box. The LT50 of 3rd instar larvae infected from insect pulp and freeze-dried insect pulp was significantly lower than that of those infected from virus suspension and freeze-dried virus suspension. There was no significant change in the insecticidal activity of HcNPV when stored for less than 12 months and insecticidal activity did not change significantly over a range of storage temperatures from - 20 to 25 °C. The best preservatives were sucrose (50%) and sodium chloride (5%), which had in LT50 values of 4.94 days and 4.97 days, respectively. [Conclusion] The highest yield of HcNPV was achieved by giving 5th instar H. cunea larvae a single feed of an artificial diet containing HcNPV inoculum at a concentration of 1x106 OB mL-1 and keeping larvae at a density of 0.05 larvae/cm² at a temperature of 20 °C. Grinding the bodies of diseased larvae into a slurry, adding sucrose (50%) or sodium chloride (5%) as adjuvants, and storing the resultant homogenate in a refrigerator at - 20 °C, allowed HcNPV to be stored for up to 12 months with no loss in insecticidal activity. [ABSTRACT FROM AUTHOR]