Interleukin-10 (IL-10) deficient dendritic cells (DCs) are potent antigen presenting cells during Chlamydia infection. To further understand the mechanism underlying this protective property in IL-10 DCs, we combined two proteomic techniques: 2DIGE and liquid chromatography mass spectrometry. We then performed western blotting on proteins from Chlamydia infected wild type (WT) and IL-10 knock out (KO) DCs. The results showed that alpha enolase (ENO1), a metabolic enzyme involved in the ninth step of glycolysis, was significantly upregulated in Chlamydia infected IL-10KO DCs compared to WT DCs. We further studied the role of ENO1 by silencing ENO1 gene using lentiviral siRNA technology. Flow cytometry, confocal microscopy, cytokine analysis, infectivity analysis and T-cell proliferation analysis were also used to determine DC function. We then analyzed the effect of the ENO1 knockdown on DC metabolism during Chlamydia stimulation. The results showed that DC maturation and activation were decreased in ENO1 knockdown DCs that were stimulated with Chlamydia compared to the Chlamydia stimulated WT DCs. In addition, pyruvate concentration decreased significantly in ENO1 knockdown DCs stimulated with Chlamydia. The mitochondrial structure of Chlamydia stimulated ENO1 knockdown DCs appeared damaged compared to the Chlamydia stimulated WT DCs. The function of ENO1 in the immune response to Chlamydia trachomatis infection is unknown. However, the results from this study indicates that ENO1 may contribute to DC metabolism and mitochondrial homeostasis which may play a role in the maturation and antigen presenting function of DCs.