In the mevalonic acid (MVA) pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is considered the first rate-limiting enzyme in isoprenoid biosynthesis. In this study, we cloned a full-length cDNA from Populus trichocarpa with an open reading frame of 1,734 bp. The deduced PtHMGR sequence contained two HMG-CoA motifs and two NADPH motifs, which exhibited homology with HMGR proteins from other species. Subsequently, truncated PtHMGR was expressed in Escherichia coli BL21 (DE3) cells, and enzyme activity analysis revealed that the truncated PtHMGR protein could catalyze the reaction of HMG-CoA and NADPH to form MVA. Relative expression analysis suggests that PtHMGR expression varies among tissues and that PtHMGR responds significantly to abscisic acid (ABA), NaCl, PEG 6000, hydrogen peroxide (H 2O 2), and cold stresses. We used polymerase chain reaction (PCR) analysis to select transgenic Nanlin 895 poplars ( Populus× euramericana cv.) and quantitative reverse-transcription PCR (qRT-PCR) to show that PtHMGR expression levels were 3- to 10-fold higher in transgenic lines than in wild-type (WT) poplars. qRT-PCR was also used to determine transcript levels of methylerythritol phosphate (MEP)-, MVA-, and downstream-related genes, indicating that overexpression of PtHMGR not only affects expression levels of MVA-related genes, but also those of MEP-related genes. We also measured the content of terpenoids including ABA, gibberellic acid (GA), carotenes, and lycopene. PtHMGR overexpression significantly increased ABA, GA, carotene, and lycopene content, indicating that PtHMGR participates in the regulation of terpenoid compound synthesis.