Overweight/obesity (OW/OB) has been associated with gut dysbiosis, changes in gastrointestinal motility and sedentary behavior, contributing to metabolic and inflammatory alterations. This systematic review aims to assess the evidence supporting the influence of physical activity and exercise on gut microbiota composition and diversity in OW/OB and was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, using MEDLINE, EMBASE, EBSCO, and Scopus databases. Risk of bias was assessed with RoB 2 for randomized controlled trials (RCTs), ROBINS-I for non-RCT, and JBI Critical Appraisal tool for cross-sectional studies. Eleven studies were selected including 476 OW/OB and 382 normal weight individuals. Seven studies included different types of exercise intervention while the other four were cross-sectional studies assessing physical activity. Results show no clear evidence of a less diverse microbiota in OW/OB. Exercise does not significantly affect alpha diversity of gut microbiota but modifies beta diversity depending on OB status. Moderate to vigorous physical activity positively associates with gut microbiota composition and short-chain fatty acid producing bacteria. These findings highlight the importance of considering gut microbiota contribution to inter-individual variability of response to obesity treatments. Modulation of gut microbiota through physical activity should be considered in the design of personalized therapeutic strategies in obesity. PROSPERO registration number: CRD42021262107.