The plant chloroplast thylakoid membrane must respond to environmental variations in light intensity to maximise the efficiency of photosynthesis and minimise photo-oxidative stress. Plants respond to changing light intensity in the short-term (seconds to minutes) through altered regulation of the structure and function of existing thylakoid components, whereas long-term acclimation (hours to days) involves changes in gene expression, protein synthesis and degradation, which modulate the composition of the thylakoid membrane. Here we have investigated the long-term changes in the thylakoid membrane composition in Arabidopsis thaliana plants acclimated to low, moderate and high light intensities using quantitative label-free proteomics in combination with a range of biochemical and spectroscopic functional analyses.