Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is presently used in physiological evaluations for visualisation of targets in organs. In the present study, MALDI-MSI was used as a visualisation technique to investigate the intestinal absorption of polyphenols. Nifedipine/phytic acid-aided MALDI-MSI was performed to visualise theaflavin-3'-O-gallate (TF3'G) and epicatechin-3-O-gallate (ECG) in the rat jejunum for 50-µM, 60-min transport experiments. Non-absorbable TF3'G was successfully visualised at the apical region, whereas absorbable ECG was detected throughout the rat jejunum. MALDI-MSI was also performed to determine the transport routes of the target metabolites. Signals corresponding to TF3'G and ECG in the membranes were diminished following treatment with inhibitors targeting the monocarboxylic acid transporter and organic anion transporting polypeptides. Enhanced visualisation of TF3'G was achieved by inhibiting efflux routes. Our findings demonstrated that the present MALDI-MSI can provide critical spatial informations on intestinal absorption of targets, by which TF3'G and ECG were incorporated into intestinal tissues, followed by efflux back to the apical compartment. In addition, MALDI-MSI analyses suggested that TF3'G was resistant to phase II metabolism during the influx/efflux processes, whereas ECG was susceptible to methylation and sulphation reactions. In conclusion, inhibitor-aided MALDI-MSI could serve as a powerful in situ visualisation technique for verifying intestinal transport routes and investigating the metabolism of penetrants.