The aim of this work was to identify S-acylated proteins in poplar. In order to achieve this, we have combined the acyl-biotin exchange method with a mass spectrometry based proteomic approach. To date, this is the only study on S-acylated proteins in a tree species. In total, we identified around 450 S-acylated proteins, the majority of which are involved in signal transduction, response to stress and transport.