Anaerobic bacteria are considered intrinsically resistant to aminoglycosides due to poor antibiotic uptake. However, many strains of the anaerobic gut pathogen Clostridioides difficile (C. difficile) carry aminoglycoside-modifying enzymes (AMEs), which are known to confer aminoglycoside resistance in other bacteria. The abundance of AMEs is not thought to be associated with aminoglycoside resistance in C. difficile given its intrinsic resistance to these antibiotics. Instead, C. difficile is hypothesised to be a resistance gene carrier from which AMEs can spread to other bacteria. Here, we show that C. difficile is unexpectedly sensitive to aminoglycosides when antibiotic susceptibility testing is performed in a medium that mimics gut conditions. Antibiotic uptake assays show that C. difficile accumulates aminoglycosides more efficiently when grown under gut-like conditions, which appears to be driven independently of the membrane proton motive force. In the presence of AMEs, C. difficile is resistant to aminoglycosides as shown by in vitro antibiotic susceptibility tests. Finally, using a C. difficile infection (CDI) mouse model, we show that aminoglycosides are effective in reducing C. difficile colonisation numbers and in enhancing survival in treated mice. We also show that aminoglycoside treatment loses efficacy when mice are infected with an AME-carrying C. difficile strain, resulting in no survival in the treated mice. Our findings have uncovered that, contrary to current thinking, C. difficile may not be intrinsically resistant to aminoglycosides since changes in the environmental conditions could significantly alter its response to this class of antibiotics. We also conclude that AMEs protect C. difficile against the lethal effects of aminoglycosides and that C. difficile is not a silent carrier of these resistance genes. More broadly, aminoglycosides may provide a promising option for adjunctive therapy in the treatment of CDI to extend the use of our ever-diminishing toolbox of antibiotics in the fight against infectious diseases.