Resveratrol is one of the most studied plant secondary metabolites owing to its numerous health benefits. It is accumulated in some plants following biotic and abiotic stress pressures, including UV-C irradiation. Polygonum cuspidatum represents the major natural source of concentrated resveratrol but the underlying mechanisms as well as the effects of UV-C irradiation on resveratrol content have not yet been documented. Herein, we found that UV-C irradiation significantly increased by 2.6-fold and 1.6-fold the resveratrol content in irradiated leaf samples followed by a dark incubation for 6 h and 12 h, respectively, compared to the untreated samples. De novo transcriptome sequencing and assembly resulted into 165,013 unigenes with 98 unigenes mapped to the resveratrol biosynthetic pathway. Differential expression analysis showed that P. cuspidatum strongly induced the genes directly involved in the resveratrol synthesis, including phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate-CoA ligase and stilbene synthase (STS) genes, while strongly decreased the chalcone synthase (CHS) genes after exposure to UV-C. Since CHS and STS share the same substrate, P. cuspidatum tends to preferentially divert the substrate to the resveratrol synthesis pathway under UV-C treatment. We identified several members of the MYB, bHLH and ERF families as potential regulators of the resveratrol biosynthesis genes.