In a variety of countries, juvenile idiopathic arthritis (JIA) is the most common cause of chronic arthritis in childhood, yet its etiology is still unknown. In recent years, etanercept, an effective inhibitor of tumor necrosis factor alpha (TNF-α), was used as an alternative in certain oligoarticular JIA patients resistant to conventional nonsteroidal antiinflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), and corticosteroid therapies, and it resulted in sustained improvement in JIA symptoms. This pilot study explores the alterations of specific panels of cytokines and protein profiles in plasma for two Taiwanese pediatric cases with diagnosed enthesitis-related arthritis (ERA), a type of JIA. The patients were studied before and after taking etanercept alone, using a high-content screening approach employing membrane-based human cytokine antibody microarray and the conventional two-dimensional gel electrophoresis (2-DE) proteomic technique. Specifically, 2-DE in combination with mass spectrometry (MALDI-MS) revealed the functional roles of plasma proteins associated with the regulation of immune responses during short-term etanercept treatment of children with ERA. Our study shows that this biotherapy improved clinical ERA manifestations through the regulation of inflammatory mediators, including several cytotoxic cellular cytokines (IL-2/IFN-γ), chemokines (MCP-1), and growth factors (GRO) that affect the expression of specific acute phase proteins such as haptoglobins, immunoglobulin A, and fibrinogen-γ chain. Meanwhile, an up-regulation of antithrombin chain I, vitamin-D binding protein (VDBP), and the various apolipoproteins was also observed after the administration of etanercept in both studied children. These results may be interpreted as the relevant predictive biomarkers of therapeutic responses to etanercept. They suggest that etanercept, which is still rarely used in Taiwan, is a viable treatment for JIA patients, without adverse health effects and increased risk of secondary infections.