In plants, plasmodesmata (PD) are cellular junctions which allow cell to cell communication. These connections between plant cells are critical for plant development and plant stress responses, diffusion of molecules, proteins and nucleic acid active transport and finally, spread of virus and fungi. PD pores exhibit a varying diameter of 20-40nm and electron microscopy observations revealed in their center a tubule of endoplasmic reticulum called the desmotubule. In this way, they establish a continuity of the cytoplasm (symplasm), the plasma membrane and the endoplasmic reticulum throughout the whole plant body. Finally, a dynamic regulation of the flux through the PD leads to a finely controlled cell to cell communication. To elucidate their structure and function, proteomic analysis of PD enriched fractions have been per- formed. The difficulty was to isolate PD from a cell wall fraction. The first protocol was established in 1995 with a cellulase treatment and according to these recommendations, the first major PD proteome was determined by the laboratory of A. Maule in 2011. Their approach was based on performing a nanoLC-MS/MS method using an LTQ-OrbitrapTM mass spectrometer. They identified 1341 proteins but after a data mining, they established that at least 34% were contaminant proteins. Furthermore, this method didn’t provide any information on protein relative abundance. In 2015, our laboratory improved PD isolation protocol from Arabidopsis thaliana suspension culture cells and a new strategy of proteomic analysis of PD enriched fraction was developed. Protein relative abundances were evaluated using a label-free method. Peptide mixture from different sub-cellular frac- tions (i.e. PD, total cell extract, plasma membrane, microsomes and cell wall) were analyzed with the Ultimate 3000 nanoLC system (Dionex) coupled to the LTQ-Orbitrap XL mass spectrometer (Thermo-Finnigan). Finally, protein enrichment ratios were calculated and allow us to establish a new refined PD proteome.