Perfluorododecanoic acid (PFDoA) is a member of the perfluoroalkyl acid (PFAA) family and has broad applications and a wide distribution in the environment. Here, we used TiO(2)-based phosphopeptide enrichment coupled with LC-MS/MS analysis to identify phosphopeptides in rat livers that were influenced by PFDoA treatment. We identified a total of 1443 unique phosphopeptides from among 769 phosphoproteins identified in normal and PFDoA-treated rat livers, 849 unique phosphorylation sites were also identified. Of these sites, 143 were considered to be novel phosphorylation sites. Many phosphoproteins were found to be associated with hepatic injuries and diseases, such as hepatotoxicity, regeneration, fatty liver, neoplasms and carcinoma. Furthermore, 25 of the identified phosphoproteins were found to be related to glycogen synthase kinase-3 (GSK3), either directly or indirectly. Western blot and qPCR results suggested that chronic PFDoA exposure inhibited insulin signal pathways and that inhibition of GSK3 might contribute to the observed increases of lipid levels in the liver.