Sedentary behavior is associated with gut microbiota dysbiosis and increased metabolic risk, yet effective microbiome-modulating strategies for individuals who cannot engage in sufficient physical activity remain underexplored. Probiotics may modulate sedentary-associated microbial alterations. We conducted a cross-sectional multi-omics comparison of physically active (PA, n = 65) and physically inactive (PI, n = 90) young adults, followed by a 6-week randomized, double-blind, placebo-controlled trial in the PI cohort (probiotic: PIP, n = 45; placebo: PIC, n = 45). At baseline, PA individuals exhibited higher α-diversity and a more favorable microbial community structure than PI participants, enriched in Bifidobacterium adolescentis, B. bifidum, Anaerostipes hadrus, and associated metabolites. A random forest classifier distinguished PA from PI with high accuracy (96.1%; area under the curve, AUC = 0.993). Following intervention, PIP subjects showed a modest shift toward an active-like microbiome: root Jensen-Shannon similarity between PIP and PA (69.3%) was significantly higher than between PIC and PA (67.8%; P < 0.001), and levels of key taxa (A. hadrus, B. bifidum) and metabolites (vanillic acid, prolylglycine) moved closer to PA. Consistently, classifier performance decreased for PIP vs. PA (AUC 0.690; accuracy 68.2%) but remained higher for PIC vs. PA (AUC 0.843; accuracy 80.0%), indicating a probiotic-associated reduction in microbiome dissimilarity. These findings suggest that probiotic supplementation induces a partial, ecologically measurable shift toward an active-like gut microbiome configuration in inactive individuals, without replicating the full physiological benefits of regular physical activity, highlighting its potential as a scalable, adjunctive strategy for microbiome health in sedentary populations.