Time course of carotenoid and membrane lipid variation during high light (HL) acclimation (about 85 𝜇mol m−2 s−1), after transfer from low light (LL) (5–10 μmol m−2 s−1), was determined in a marine Synechococcus strain. High-performance liquid chromatography (HPLC) coupled to diode array detector (DAD) or electrospray ionization mass spectrometry (ESI-MS) was used for compound separation and detection. Myxoxanthophyll rose within a time interval of 8 h to 24 h after the onset of exposure to HL. 𝛽-carotene content started to decrease after 4 h of the onset of exposure to HL. Zeaxanthin content rose with exposure to HL, but it was only significant after 24 h of exposure. Carotenoid changes are in agreement with a coordinated activity of the enzymes of the myxoxanthophyll biosynthetic pathway, with no rate-limiting intermediate steps. Lipid analysis showed all species with a C18:3/C16:0 composition increased their content, the changes of PG(18:3/16:0) and MGDG(18:3/16:0) being primarily significant. Major lipid changes were also found to occur within 24 h. These changes might suggest reduction and reorganization of the thylakoid membrane structure. Hypotheses are also drawn on the role played by lipid molecule shape and their possible effect in membrane fluidity and protein accommodation.