We investigated the relationship between aqueous cytokine levels, changes in ocular blood flow, and morphological and functional improvements after intravitreal ranibizumab injection (IRI) in treatment-naïve eyes with center-involving diabetic macular edema (DME). Thirty-three eligible patients with DME (33 eyes) were recruited. At the first IRI, we collected a sample of aqueous humor from each eye and measured levels of the cytokines/chemokines. Mean blur rate (MBR) was used to evaluate retinal and choroidal flow by laser speckle flowgraphy at the time of the first IRI and 1 month later. One month after IRI, both retinal and choroidal MBR had significantly decreased from baseline. The reduction ratio of the retinal MBR was significantly correlated with aqueous levels of monocyte chemotactic protein (MCP)-1 and interleukin-8, and with reduction of central macular thickness, but not with improvement of best corrected visual acuity. The reduction ratio of choroidal MBR showed no statistical correlation with any cytokine levels or changes in clinical parameters. We conclude that IRI reduces both retinal and choroidal blood flow in treatment-naïve DME. Reduction of retinal blood flow correlated with regression of morphological pathology, which is regulated by the initial aqueous levels of some cytokines.