Background/Aims: We investigated gut flora characteristics in patients with functional constipation (FC) and influences of short-term treatment with VSL#3 probiotic on flora and symptom improvement. Methods: Thirty patients fulfilling Rome III criteria for FC and 30 controls were enrolled. Fecal samples were obtained before and after VSL#3 intake (one sachet twice daily for 2 weeks) and flora were examined by quantitative real-time polymerase chain reaction (qRT-PCR). Symptom changes were also investigated. Results: The fold differences in Bifidobacterium and Bacteroides species were significantly lower in feces from FC, compared to in controls (P = 0.030 and P = 0.021). After taking VSL#3, the fold differences in Lactobacillus, Bifidobacterium and Bacteroides species increased in controls (P = 0.022, P = 0.018, and P = 0.076), but not in FC. Mean Bristol scores and complete spontaneous bowel movements (CSBMs)/week increased significantly in FC after ingesting VSL#3 (both P < 0.001). Relief of subjective CSBM frequency, stool consistency and abdominal bloating were reported in 70%, 60%, and 47% of patients. After VSL#3 cessation, 44.4% of patients with symptom improvement experienced constipation recurrence mostly within one month. Conclusions: Bifidobacterium and Bacteroides species might be quantitatively altered in FC. A short-term VSL#3 treatment can improve clinical symptoms of FC. Further studies are needed to investigate VSL#3's additional effects beyond altering gut flora to allevate constipation.