Most rumen bacteria are uncultured, making their niche hard to identify. Fluorescent substrates could potentially identify the substrate preferences and the niche of these uncultured bacteria, but uptake of these substrates has not been tested with mixed rumen bacteria. Our objective was to determine if a fluorescent analog of glucose (2-NBDG) would be taken up by mixed bacteria from the rumen. A second objective was to determine if we could separate cells taking up 2-NBDG by using fluorescence-activated cell sorting. We prepared mixed bacteria from rumen fluid by centrifugation, incubated them in 2-NBDG (0 to 100 μM) for up to 60 s, and monitored uptake of 2-NBDG with fluorimetry. We found mixed bacteria took up 2-NBDG, and they did so with a maximum velocity (V_max) of 0.180 (0.05 SEM) nmol mg protein⁻¹ min⁻¹ and Michaelis constant (K_m) of 6.37 (SEM 4.86) μM (n = 3). We confirmed that cells took up 2-NBDG by using flow cytometry, which revealed that up to 18.5% cells were positive after incubating them in 100 μM 2-NBDG for 5 min. Positive cells could be separated with fluorescence-activated cell sorting, and post-sort analysis revealed 94% of sorted positive cells were in fact positive. Work is ongoing to (1) optimize cell sorting, (2) sequence sorted cells for identification, and (3) synthesize and test uptake of other glucose analogs. Our work supports that 2-NBDG, in combination with other analogs, could be used to identify which bacteria consume which substrates, helping define what role uncultured bacteria play in the host.