The variations in the membrane proteome of tomato fruit pericarp during ripening have been investigated by mass spectrometry-based label-free proteomics. Mature green (MG30) and red ripe (R45) stages were chosen because they are pivotal in the ripening process: MG30 corresponds to the end of cellular expansion, when fruit growth has stopped and fruit starts ripening, whereas R45 corresponds to the mature fruit. Protein patterns were markedly different: among the 1315 proteins identified with at least two unique peptides, 145 significantly varied in abundance in the process of fruit ripening. The subcellular and biochemical fractionation resulted in GO term enrichment for organelle proteins in our dataset, and allowed the detection of low-abundance proteins that were not detected in previous proteomic studies on tomato fruits. Functional annotation showed that the largest proportion of identified proteins were involved in cell wall metabolism, vesicle-mediated transport, hormone biosynthesis, secondary metabolism, lipid metabolism, protein synthesis and degradation, carbohydrate metabolic processes, signalling and response to stress.