Mulberry leaves (Morus alba L.) are of high medicinal value in traditional Chinese medicine with chlorogenic acid (CGA) as its major biologically active constituent. Mulberry leaves require that they be harvested after frost; previous studies have shown CGA accumulation significantly increased after frost. However, the molecular mechanism of how frost changes the CGA content in mulberry leaves is unclear. Additionally, the mechanism of CGA biosynthesis and key genes in mulberry leaves are not well-understood. In this study, transcriptome sequencing was performed on two mulberry leaf samples with different CGA contents (before and after frost). Fifty-eight genes were annotated in the CGA biosynthetic pathway. Compared to those in pre-frost mulberry leaves, 12 and 5 genes were upregulated and downregulated, respectively, in post-frost leaves. Correlation analysis showed that the expression levels of four genes were significantly positively correlated with CGA content, including those encoding phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, hydroxycinnamoylCoA shikimate/quinate hydroxycinnamoyltransferase (HCT), and coumaroyl quinate/shikimate 3'-hydroxylase, and may be key genes in the CGA biosynthetic pathway. We cloned MaHCT4 (GenBank accession no. MH476577) from mulberry leaves. Multiple sequence alignment suggested that MaHCT4 contains the conserved domains HXXXD and DFGWG. Enzymatic assays indicated that MaHCT4 catalyzes the formation of p-coumaroyl shikimic acid, p-coumaroyl quinic acid, and CGA. The K-m values of quinic acid and shikimic acid were 10 +/- 1.0 and 31 +/- 1.7 mu M, respectively, suggesting that MaHCT4 favored quinic acid over shikimic acid as its acyl acceptor. Using quinic acid as an acyl acceptor, MaHCT4 showed a preference for p-coumaroyl-CoA over caffeoyl-CoA. Our results provide insight into the molecular mechanism of how frost alters the CGA content and roles of key genes involved in the CGA biosynthetic pathway in mulberry leaves.