Valeriana officinalis (valerian) is a popular medicinal plant in North America and Europe. Its root extract is commonly used as a mild sedative and anxiolytic. Valerenic acid, a C15 sesquiterpenoid, has been suggested as the active ingredient responsible for the sedative effect. Recently, medical uses of valerenic acid as anti-depressant and anti-inflammatory drugs were suggested due to its affinity for the γ-aminobutyric acid type A (GABAA) receptor as an agonist and its inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, respectively. Despite its importance, biochemistry of valerenic acid in valerian remains unknown. To identify the first committed enzymatic step in valerenic acid biosynthesis, next-generation sequencing (Roche 454 titanium) was used to generate ~1 million transcript reads from valerian root. Subsequently, three cDNAs for sesquiterpene synthases (VoTPS1/2/3) were identified and their corresponding recombinant enzymes were purified. Three recombinant enzymes efficiently catalyze the synthesis of valerena-4,7(11)-diene, germacrene C/D, and drimenol, respectively, based on the spectral match in the mass spectrometry library. Additional structural analyses using GC-MS and 13C-NMR spectrometry in comparison to a semi-synthesized standard confirmed the chemical identity of valerena-4,7(11)-diene. This is the first report of valerena-4,7(11)-diene and drimenol synthases, and the biosynthetic mechanisms of these two products from the substrate, farnesyl diphosphate, were proposed.