Dietary fiber (DF)-based interventions are crucial in establishing a health-promoting gut microbiota. However, directly investigating DFs’ in vivo interactions with intestinal bacteria remains challenging due to the lack of suitable tools. Here, we develop an in vivo metabolic labeling-based strategy, which enables not only imaging and identifying the bacteria that bind with specific DF in the intestines, but also quantifying DF's impact on their metabolic status. Four DFs, including galactan, rhamnogalacturonan and two inulins, are fluorescently derivatized and used for in vivo labeling to visually record DFs’ interactions with gut bacteria. The subsequent cell-sorting, 16S rDNA sequencing, and fluorescence in situ hybridization identify the taxa that bind each DF. We then select a DF-binding species newly identified herein and verify its DF-catabolizing capability in vitro. Furthermore, we find that the indigenous metabolic status of Gram-positive bacteria, whether inulin-binders or not, is significantly enhanced by the inulin supplement. This trend is not observed in Gram-negative microbiota, even for the inulin-binders, demonstrating the ability of our methods in differentiating the primary, secondary DF-degraders from cross-feeders, a question that is difficult to answer by using other methods. Our strategy provides a novel chemical biology tool for deciphering the complex DF-bacteria interactions in the gut.