A two-color vibrational imaging technique for simultaneously mapping glucose uptake and incorporation activity inside single living cells is reported. Heterogeneous patterns of glucose metabolism are directly visualized from the ratiometric two-color images for various cell types, cells undergoing epithelia-to-mesenchymal transitions and live mouse brain tissues. The two-color imaging of glucose metabolism here demonstrates the development of multi-functional vibrational probes for multicolor imaging of cellular metabolism.