Polyprenols are ubiquitous isoprenoid compounds that accumulate in large quantities in plant photosynthetic tissues. While our knowledge of polyprenol biochemistry is constantly expanding, the regulation of their biosynthesis as well as the molecular basis of their cellular action are still poorly understood. In Arabidopsis, the polyprenols Pren-9, −10 and −11, synthesized by cis -prenyltransferase 7 (CPT7), are localized in plastidial membranes and affect the photosynthetic performance of chloroplasts. In this report we present evidence that plastidial polyprenols are among the major constituents of thylakoid membranes. Disturbances in polyprenol level, caused by alterations in CPT7 expression, change chloroplast ultrastructure, affect aggregation of LHCII complexes and modulate non-photochemical quenching (NPQ). Moreover, we show that Arabidopsis responds to high temperature by upregulating expression of CPT7 and increasing the accumulation of CPT7-derived polyprenols. These heat-induced changes in polyprenol biosynthesis are mediated by Heat Shock Transcription Factors of the HSFA1 family, the master regulators of heat stress response. Collectively, results presented in this report bring us closer to understanding the mechanisms by which polyprenols affect plant physiology and provide an additional link between chloroplast biology and plant responses to changing environmental conditions. One sentence summary: Heat Shock Transcription Factors induce biosynthesis of polyprenols - isoprenoid compounds that affect the organization and function of chloroplasts.