This study aimed to investigate gastrointestinal tolerability, treatment persistence and iron status markers in patients with iron-deficiency anemia (IDA) who received oral iron replacement therapy (IRT) with versus without concomitant Lactobacillus plantarum 299v (L. plantarum 299v) probiotic supplementation. A total of 295 patents with newly diagnosed IDA were randomly assigned to receive either IRT alone (n=157, IRT-only group) or IRT plus L. plantarum 299v (n=138, IRT-Pro group) in this prospective randomized non-placebo-controlled study (ClinicalTrials.gov Identifier: NCT06521879). Gastrointestinal intolerance symptoms (at baseline, within the first 30 days of IRT and at any time during 3-month IRT), serum hemoglobin levels (at baseline and 3rd month of IRT) and iron status markers (at baseline and 3rd month of IRT) were recorded. IRT-Pro group, when compared to IRT-only group, experienced significantly lower rates of gastrointestinal intolerance over the course of IRT (13.0% vs. 46.5%, p<0.001) and treatment discontinuation within the first 30 days (3.6% vs. 15.9%, p<0.001). At 3rd month of therapy, IRT-Pro vs. IRT-only group had significantly higher serum levels for iron (76.0(51.0-96.0) vs. 60.0(43.0-70.0) µg/dL, p<0.001) and transferrin saturation (20.1(12.5-28.5) vs. 14.5(10.5-19.0) %, p<0.001) and higher change from baseline hemoglobin (0.9(0.3-1.3) vs. 0.4(-0.1-1.1) g/dL, p<0.001) levels. Use of L. plantarum 299v probiotic supplementation during the first 30 days of IRT in IDA patients significantly reduces the gastrointestinal burden of IRT (particularly abdominal pain and bloating), the likelihood of intolerance development (by ∼3 times) and treatment discontinuation (by∼5 times), as accompanied with improved serum hemoglobin levels and serum iron markers.