Protein palmitoylation is an essential post-translational lipid modification of proteins, and reversibly orchestrates a variety of cellular processes. Identification of palmitoylated proteins with their sites is the foundation for understanding molecular mechanisms and regulatory roles of palmitoylation. Contrasting to the labor-intensive and time-consuming experimental approaches, in silico prediction of palmitoylation sites has attracted much attention as a popular strategy. In this work, we updated our previous CSS-Palm into version 2.0. An updated clustering and scoring strategy (CSS) algorithm was employed with great improvement. The leave-one-out validation and 4-, 6-, 8- and 10-fold cross-validations were adopted to evaluate the prediction performance of CSS-Palm 2.0. Also, an additional new data set not included in training was used to test the robustness of CSS-Palm 2.0. By comparison, the performance of CSS-Palm was much better than previous tools. As an application, we performed a small-scale annotation of palmitoylated proteins in budding yeast. The online service and local packages of CSS-Palm 2.0 were freely available at: http://bioinformatics.lcd-ustc.org/css_palm.