Despite the importance of glucose as an energy source for marginal cells of the cochlear stria vascularis, no direct evidence of the glucose uptake into these cells has been demonstrated. In this study, we investigated the glucose pathway in strial marginal cells using a fluorescent tracer method combined with immunohistochemistry. For glucose imaging, a non-metabolizable fluorescent glucose analog, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG), was intravenously injected into mice. After 6-NBDG fluorescent signals in cochlear sections were observed, the same sections were processed for immunohistochemistry to identify marginal cells. This showed that 6-NBDG fluorescent signals had passed into the cytosol of marginal cells via Na+/K+-ATPase-positive basolateral membranes. These results provide direct physiological evidence of the glucose uptake into epithelial strial marginal cells. This combined method can help in examining the relationship between target molecules and functional proteins using the same tissue samples.