Background: Obesity and type 2 diabetes mellitus (T2DM) are characterized by underlying low-grade chronic inflammation. Metformin has been used as the first line of therapy in T2DM as it decreases hepatic glucose production and glucose intestinal absorption, enhances insulin sensitivity and weight loss, and is known to ameliorate inflammation. The mechanisms through which metformin exerts its effect remain unclear. Proteomics has emerged as a unique approach to explore the biological changes associated with diseases, including T2DM. It provides insight into the circulating biomarkers/mediators which could be utilized for disease screening, diagnosis, and prognosis. Methods: This study evaluated the proteomic changes in obese (Ob), obese diabetics (OD), and obese diabetic patients on metformin (ODM) using a 2D DIGE MALDI-TOF mass spectrometric approach. Results: Significant changes in sixteen plasma proteins (15 up and 1 down, ANOVA, p ≤ 0.05; fold change ≥ 1.5) were observed in the ODM group when compared to the Ob and OD groups. Bioinformatic network pathway analysis revealed that the majority of these altered plasma proteins are involved in distinct pathways involving acute-phase response, inflammation, and oxidative response and were centered around HNF4A, ERK, JNK, and insulin signaling pathways. Conclusions: Our study provides important information about the possible biomarkers altered by metformin treatment in obese patients with and without T2DM. These altered plasma proteins are involved in distinct pathways involving acute-phase response, inflammation, and oxidative response and were centered around HNF4A, ERK, JNK, and insulin signaling pathways. The presented proteomic profiling approach may help in identifying potential biomarkers/mediators affected by metformin treatment in T2DM and inform the understanding of metformin's mechanisms of action.