INTRODUCTION: Constipation is one of the common non-motor symptoms in Parkinson's disease that significantly impacts patient quality of life. Probiotics supplementation may improve constipation symptoms, but its effect on the gut microbiota population is unclear. METHODS: In this randomized controlled study, 46 PD patients with constipation according to Rome Ⅲ criteria were recruited. The number of complete bowel movements per week, degree of defecation effort, Bristol stool Scale (BSS), Patient Assessment of Constipation symptom (PAC-SYM) and Patient assessment of constipation quality of life questionnaire (PAC-QOL) were collected pre- and post-intervention to evaluate the constipation symptoms. In addition, fresh feces of subjects before and after intervention and healthy controls were collected for 16s rRNA gene V3-V4 region sequencing to compare bacterial flora differences. RESULTS: Compared with the control group, the treatment group increased the average number of complete bowel movements per week (1.09 ± 1.24 vs. 0.04 ± 0.64, P < 0.001). Probiotics supplementation reduced the BSS score (0.65 ± 0.93 vs. -0.17 ± 0.94, P = 0.004), PAC-SYM score (4.09 ± 6.31 vs. -1.83 ± 4.14, P < 0.001), PAC-QOL score (10.65 ± 16.53 vs. 0.57 ± 12.82, P = 0.042), and degree of defecation effort score (1.00 ± 0.80 vs. 0.00 ± 0.30, P < 0.001). The improvement rate of constipation in the probiotics group was significantly higher than that in the control group (52.2% vs. 8.7%, P = 0.001). PD patients showed intestinal flora disorders compared to healthy controls. After 12 weeks of probiotics treatment, g_Christensenella_sp._Marseille-P2437 significantly increased, while g_Eubacterium_oxidoreducens_group, g_Eubacterium_hallii_group and s_Odoribacter_sp._N54.MGS-14 decreased (p < 0 0.05). CONCLUSIONS: Probiotics treatment can effectively improve the constipation symptoms of PD patients and positively affected the gut microbiota.