The redox state of the plastoquinone (PQ) pool is a known sensor for retrograde signaling. In this paper, we asked, “does the redox state of the PQ pool modulate the saturation state of thylakoid lipids?” Data from fatty acid composition and mRNA transcript abundance analyses suggest a strong connection between these two aspects in a model marine diatom. Fatty acid profiles of Phaeodactylum tricornutum exhibited specific changes when the redox state of the PQ pool was modulated by light and two chemical inhibitors [3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)]. Data from liquid chromatography with tandem mass spectrometry (LC–MS/MS) indicated a ca. 7–20% decrease in the saturation state of all four conserved thylakoid lipids in response to an oxidized PQ pool. The redox signals generated from an oxidized PQ pool in plastids also increased the mRNA transcript abundance of nuclear-encoded C16 fatty acid desaturases (FADs), with peak upregulation on a timescale of 6 to 12 h. The connection between the redox state of the PQ pool and thylakoid lipid saturation suggests a heretofore unrecognized retrograde signaling pathway that couples photosynthetic electron transport and the physical state of thylakoid membrane lipids.