Mechanisms of abnormal protein phosphorylation that regulate cell invasion and metastasis in pancreatic cancer remain obscure. In this study, we used high-throughput phosphorylation array to test two pancreatic cancer cell lines (PC-1 cells with a low, and PC-1.0 cells with a high potential for invasion and metastasis). We noted that a total of 57 proteins revealed a differential expression (fold change ≥ 2.0). Six candidate proteins were further validated by western blot with results found to be accordance with the array. Of 57 proteins, 32 up-regulated proteins (e.g. CaMK1-α and P90RSK) were mainly involved in ErbB and neurotrophin signaling pathways as determined using DAVID software, while 25 down-regulated proteins (e.g. BID and BRCA1) were closely involved in apoptosis and p53 signaling pathways. Moreover, four proteins (AKT1, Chk2, p53 and P70S6K) with different phosphorylation sites were found, not only among up-regulated, but also among down-regulated proteins. Importantly, specific phosphorylation sites can affect cell biological functions. CentiScaPe software calculated topological characteristics of each node in the protein-protein interaction (PPI) network: we found that AKT1 owns the maximum node degrees and betweenness in the up-regulation protein PPI network (26 nodes, average path length: 1.89, node degrees: 6.62±4.18, betweenness: 22.23±35.72), and p53 in the down-regulation protein PPI network (17 nodes, average path length: 2.04, node degrees: 3.65±2.47, betweenness: 16.59±29.58). In conclusion, the identification of abnormal protein phosphorylation related to invasion and metastasis may allow us to identify new biomarkers in an effort to develop novel therapeutic drug targets for pancreatic cancer treatment.