Background: Diarrhea is one of the dose limiting toxicity of irinotecan. SN-38 is main irinotecan metabolite responsible for diarrhea development. SN-38 is excreted in glucuronidated form into the intestine. Due to the bacterial enzyme beta-D-glucuronidase in intestinal lumen it is deconjugated and in this form causes direct damage to the intestinal mucosa associated with the development of diarrhea. This study aimed to determine the effectiveness of the probiotics in the prevention of irinotecan induced diarrhea due to reduction of intestinal beta-D-glucuronidase activity. Methods: Between January 2011 and December 2013, 46 patients with colorectal cancer starting a new line of irinotecan based therapy were included. 5-flurouracil/capecitabine along with irinotecan was administered to 26 (56.5%) patients and 22 (47.8%) patients received biological therapy as well. Patients were randomized 1:1 to probiotics (PRO) or placebo (PLA). Probiotic formula Colon Dophilus, was administered at a dose of 10x 10⁹CFU of bacteria tid, orally for 12 weeks of chemotherapy. Primary endpoint was incidence of grade 3/4 diarrhea. The study was prematurely terminated due to slow accrual, when 46 of 220 planned patients were accrued. Herein, we report final analysis of the study. Results: 23 patients were randomized to PRO and 23 patients to PLA. Administration of probiotics compared to placebo led to a reduction in the incidence of severe diarrhea of grade 3 or 4 (0% for PRO vs. 17.4% for PLA, p = 0.11), as well as reduction of the overall incidence of diarrhea (39.1% for PRO vs. 60.9% for PLA, p = 0.24) and incidence of enterocolitis (0% for PRO vs. 8.7% for PLA). Patients on PRO used less loperamid compared to PLA (mean duration of loperamid use: 4.5 days for PRO vs. 10.4 days for PLA, p = 0.45; mean number of loperamid tablets: 5.9 for PRO vs. 37.7 for PLA, p = 0.49). There was no infection caused by probiotic strains recorded. Conclusions: Administration of probiotics in patients with colorectal cancer treated with irinotecan-based chemotherapy is safe and could lead to a reduction in the incidence and severity of gastrointestinal toxicity. Phase III clinical trials with adequate sample size are warranted. Clinical trial information: NCT01410955.