Purpose: This study aimed to investigate changes in cytokine levels after intravitreal bevacizumab injection in patients with chronic central serous chorioretinopathy (CSC). Methods: In a prospective interventional trial, 12 eyes from 12 patients with chronic CSC and six eyes from six patients who underwent cataract surgery were included as controls. Patients diagnosed as with CSC received a single intravitreal injection of bevacizumab (1.25 mg/0.05 mL). Aqueous humor samples were collected from the patients and controls. Best-corrected visual acuity and foveal thickness were evaluated, and aqueous samples were obtained before and 4 weeks after injection. The aqueous concentrations of interleukin (IL)-6, IL-8, interferon-induced protein (IP)-10, monocyte chemotactic protein (MCP)-1, platelet-derived growth factor (PDGF)-AA, and vascular endothelial growth factor (VEGF) were measured using a multiplex bead assay. Results: After injection, the foveal thickness decreased significantly from 328.08 μm (range, 210–477 μm) to 283.91 μm (range, 168–356 μm; p = 0.048), but the best-corrected visual acuity was not significantly different (p = 0.066). The aqueous levels of IL-8 increased significantly from 3.3 pg/mL (range, 1.5–8.3 pg/mL) to 4.7 pg/mL (range, 2.2–11.6 pg/mL) at 4 weeks after the injection (p = 0.046). The aqueous levels of VEGF decreased significantly from 31.4 pg/mL (range, 17.0–53.3 pg/mL) to 15.2 pg/mL (range, 7.7–21.5 pg/mL; p < 0.01). No significant changes in levels of IL-6 (p = 0.455), IP-10 (p = 0.055), MCP-1 (p = 0.076), and PDGF-AA (p = 0.339) were noted 4 weeks after injection. Conclusions In this study we found intravitreal bevacizumab injection decreased VEGF and increased IL-8 in the eyes of patients with chronic CSC. This study suggests the possibility that the pathogenesis of CSC may be related to abnormal circulation of the choroidal blood vessels through VEGF and IL-8 cytokine level changes.