In eukaryotes, dolichols (C70-120) play indispensable roles as glycosyl carrier lipids in the biosynthesis of glycoproteins on endoplasmic reticulum. In addition to dolichols, seed plants have other types of Z,E-mixed polyisoprenoids termed ficaprenol (tri-trans,poly-cis-polyprenol, C45-75) and betulaprenol (di-trans,poly-cis-polyprenol, C30-45 and C≥70) in abundance. However, the physiological significance of these polyprenols has not been elucidated because of limited information regarding cis-prenyltransferases (cPTs) which catalyze the formation of the structural backbone of Z,E-mixed polyisoprenoids. In the comprehensive identification and characterization of cPT homologues from Arabidopsis thaliana, AtHEPS was identified as a novel cis,trans-mixed heptaprenyl diphosphate synthase. AtHEPS heterologously expressed in Escherichia coli catalyzed the formation of C35 polyisoprenoid as a major product, independent of the chain lengths of all-trans allylic primer substrates. Kinetic analyses revealed that farnesyl diphosphate was the most favorable for AtHEPS among the allylic substrates tested suggesting that AtHEPS was responsible for the formation of C35 betulaprenol. AtHEPS partially suppressed the phenotypes of a yeast cPT mutant deficient in the biosynthesis of dolichols. Moreover, in A. thaliana cells, subcellular localization of AtHEPS on the endoplasmic reticulum was shown by using green fluorescent protein fused proteins. However, a cold-stress-inducible expression of AtHEPS suggested that AtHEPS and its product might function in response to abiotic stresses rather than in cell maintenance as a glycosyl carrier lipid on the endoplasmic reticulum.