Probiotics, classified into monostrain, multistrain and multispecies formulations, have been suggested as effective interventions for alleviating constipation related symptoms by modulating gut motility and microbiota composition. However, the comparative efficacy of these probiotics has not been systematically evaluated. This study presents a systematic review and meta-analysis of randomized controlled trials (RCTs) to assess the effects of monostrain and multispecies probiotics on constipation symptoms and gut microbiota. A total of 15 eligible RCTs were included in the analysis. Probiotic supplementation significantly improved spontaneous bowel movements (standardized mean difference (SMD) = 0.60, 95% confidence interval (CI) (0.27, 0.94), P = 0.0005) and stool consistency (SMD = 0.25, 95% CI (0.03, 0.48), P = 0.03), but had no significant effect on gut transit time (SMD = −0.04, 95% CI (−0.34, 0.27), P = 0.82) or quality of life (QoL) scores (P = 0.42). Importantly, no superior efficacy of multispecies probiotics over monostrain formulations was observed, except for a modest improvement in QoL (P < 0.00001). Additionally, probiotics had a measurable impact on gut microbiota composition, although changes in microbial diversity were not consistently correlated with clinical outcomes. Collectively, these findings suggest that probiotics can be effective in alleviating specific constipation-related symptoms, but the choice between monostrain and multispecies formulations should be based on strain-specific characteristics rather than the number of strains or species included. Additionally, this study underscored the need for future research into optimizing strain combinations and understanding probiotic-host interactions to enhance therapeutic outcomes.