AbstractAntimicrobial resistance poses a substantial threat to human health. The gut microbiome is considered a reservoir for potential spread of resistance genes from commensals to pathogens, termed the gut resistome. The impact of probiotics, commonly consumed by many in health or in conjunction with the administration of antibiotics, on the gut resistome is elusive. Reanalysis of gut metagenomes from healthy antibiotics-naïve humans supplemented with an 11-probiotic-strain preparation, allowing direct assessment of the gut resistome in situ along the gastrointestinal (GI) tract, demonstrated that probiotics reduce the number of antibiotic resistance genes exclusively in the gut of colonization-permissive individuals. In mice and in a separate cohort of humans, a course of antibiotics resulted in expansion of the lower GI tract resistome, which was mitigated by autologous faecal microbiome transplantation or during spontaneous recovery. In contrast, probiotics further exacerbated resistome expansion in the GI mucosa by supporting the bloom of strains carrying vancomycin resistance genes but not resistance genes encoded by the probiotic strains. Importantly, the aforementioned effects were not reflected in stool samples, highlighting the importance of direct sampling to analyse the effect of probiotics and antibiotics on the gut resistome. Analysing antibiotic resistance gene content in additional published clinical trials with probiotics further highlighted the importance of person-specific metagenomics-based profiling of the gut resistome using direct sampling. Collectively, these findings suggest opposing person-specific and antibiotic-dependent effects of probiotics on the resistome, whose contribution to the spread of antimicrobial resistance genes along the human GI tract merit further studies.