Faecal sample collection is crucial for gut microbiome research and its clinical applications. However, while patients and healthy volunteers are routinely asked to provide stool samples, their attitudes towards sampling remain largely unknown. Here, we investigate the attitudes of 780 Dutch patients, including participants in a large Inflammatory Bowel Disease (IBD) gut microbiome cohort and population controls, in order to identify barriers to sample collection and provide recommendations for gut microbiome researchers and clinicians. We sent questionnaires to 660 IBD patients and 112 patients with other disorders who had previously been approached to participate in gut microbiome studies. We also conducted 478 brief interviews with participants in our general population cohort who had collected stool samples. Statistical analysis of the data was performed using R. 97.4% of respondents reported that they had willingly participated in stool sample collection for gut microbiome research, and most respondents (82.9%) and interviewees (95.6%) indicated willingness to participate again, with their motivations for participating being mainly altruistic (57.0%). Responses indicated that storing stool samples in the home freezer for a prolonged time was the main barrier to participation (52.6%), but clear explanations of the sampling procedures and their purpose increased participant willingness to collect and freeze samples (P = 0.046, P = 0.003). To account for participant concerns, gut microbiome researchers establishing cohorts and clinicians trying new faecal tests should provide clear instructions, explain the rationale behind their protocol, consider providing a small freezer and inform patients about study outcomes. By assessing the attitudes, motives and barriers surrounding participation in faecal sample collection, we provide important information that will contribute to the success of gut microbiome research and its near-future clinical applications.