Sickle cell disease (SCD) is a blood illness occurs due to point mutation in the β-globin gene and leads to serious health problems. The aim of the study was to assess probiotic Lactobacillus plantarum F4’s (L. plantarum F4) capacity to induce fetal hemoglobin (HbF) synthesis and anti-sickling effects in sickle erythrocytes isolated from individuals with sickle cell disease. Erythrocytes were isolated from 15 SCD patients and 5 controls blood samples. Erythrocytes were treated with L. plantarum F4 culture supernatant (CS) at concentrations of 30% and 60% (v/v) for up to 120 minutes, following lysozyme treatment to purify the supernatant. The Emmel test was conducted by microscopic observation after treating the samples with different doses and durations of L. plantarum F4 (CS). HPLC was utilized to assess the HbF levels in patients, while normal saline and hydroxyurea served as negative and positive controls. Statistical analyses, including ANOVA and Student’s t-test, were conducted to compare treatment effects. Results demonstrated that at a concentration of 30% (v/v) for 120 minutes, L. plantarum F4 (CS) significantly decreased sickling of SCD erythrocytes (p < 0.0001) and was equivalent to the common medication hydroxyurea. Additionally, compared to the negative control, L. plantarum F4 (CS) considerably raised the HbF% in the erythrocytes of SCD patients (p < 0.0001). For the first time, our study suggests that L. plantarum F4 significantly enhance HbF and anti-sickling activity in sickled erythrocytes, in vitro and can be further explored for in vivo animal model study and developing therapeutics for SCD.