Objective To investigate the changes of protein phosphorylation in peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (AS) and its potential regulatory role in AS. Methods PBMCs were obtained from 20 cases of AS without treatment, 15 cases of AS with drug treatment, and 20 matched healthy controls. Protein extraction, trypsin digestion, phosphorylated peptide enrichment and mass spectrometric analyses were performed. AS-related phosphorylated proteins were screened by bioinformatics analysis, and the changes of expression levels of these proteins after treatment were analyzed. Results A total of 1561 significantly differential phosphorylated peptides were detected, of which 756 peptides from 472 proteins were up-regulated, and 805 from 363 proteins were down-regulated. GO enrichment analysis showed that the proteins with altered phosphorylation were mainly involved in biological processes such as neutrophil activation and platelet aggregation. The enrichment analysis of KEGG pathway showed infection, platelet activation, T cell receptors and B cell receptor signaling pathways might be closely related to AS. Conclusion This study systematically depicted the change of protein phosphorylation in PBMCs of AS patients, providing important information to understand the pathogenesis and disease progression mechanism of AS.