Glucose plays a central role in the cellular energy metabolism. Malignant tumors exhibit an elevated rate of glycolysis over normal tissues. In this study, two near-infrared fluorescent dyes, Cypate and ICG-Der-02, with different water solubility, were conjugated to 2-amino-2-deoxy-d-glucose (2DG) to form Cypate-2DG and ICG-Der-02-2DG, respectively, for NIR fluorescent imaging of tumors in nude mice. The clear routes and tumor targeting abilities of the two NIR fluorescent 2DG probes were compared. Results showed that ICG-Der-02-2DG with higher hydrophilicity was cleared faster by kidneys than the more lipophilic Cypate-2DG. Cypate-2DG had slower but stronger tumor targeting ability compared with ICG-Der-02-2DG. To investigate the correlation between the targeting ability of the probe and the glucose transporter (GLUT1) expression levels of cancer cells, the accumulation of Cypate-2DG in tumors was assessed in MCF-7/estradiol, U87MG, MCF-7 and MDA-MB-435 tumor xenografts, which express different levels of GLUT1. The results show that both Cypate-2DG and ICG-Der-02-2DG possess tumor targeting ability on all the tumors examined, with a proportional correlation to GLUT1 expression. The findings demonstrate the broad applicability of these molecular probes for optical imaging of tumors and glucose-related pathologies. Copyright © 2012 John Wiley & Sons, Ltd.