This study aimed to investigate gastrointestinal tolerability, treatment persistence and iron status markers in patients with iron deficiency anaemia (IDA) who received oral iron replacement therapy (IRT) with v. 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 randomised non-placebo-controlled study (ClinicalTrials.gov Identifier: NCT06521879). Gastrointestinal intolerance symptoms (at baseline, within the first 30 d of IRT and at any time during 3-month IRT), serum Hb 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 with IRT-only group, experienced significantly lower rates of gastrointestinal intolerance over the course of IRT (13·0 % v. 46·5 %, P < 0·001) and treatment discontinuation within the first 30 d (3·6 % v. 15·9 %, P < 0·001). At 3rd month of therapy, IRT-Pro v. IRT-only group had significantly higher serum levels for iron (76·0 (51·0–96·0) v. 60·0(43·0–70·0) µg/dl, P < 0·001) and transferrin saturation (20·1 (12·5–28·5) v. 14·5 (10·5–19·0) %, P < 0·001) and higher change from baseline Hb (0·9 (0·3–1·3) v. 0·4 (–0·1–1·1) g/dl, P < 0·001) levels. Use of L. plantarum 299v probiotic supplementation during the first 30 d 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 by improved serum Hb levels and serum iron markers.