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Diabetic nephropathy (DN) is a major microvascular complication of diabetes mellitus (DM) driven by immune activation and chronic inflammatory cascades. Human regulatory B (Breg) cells modulate immune homeostasis and are downregulated in glomerulonephritis. We aim to evaluate B cell subsets counts in type 2 DM patients with and without nephropathy in comparison to normal control individuals. Sixty participants comprising twenty-four patients with type 2 DM without kidney injury, twenty-four patients with DN diagnosed via KDIGO guidelines, and twelve matched healthy controls underwent clinical, biochemical, and flow cytometric evaluations. CD19+ B cell subsets count, including Breg cells (CD19+CD24hiCD38hi) and memory B cell counts (CD19+CD24+CD38-) were quantified using multi-parametric flow cytometry. B cell count was significantly lower in patients with DN compared to DM and healthy control groups. Moreover, Breg and memory B cell counts were significantly lower in the DN group compared to the DM group. However, no significant difference was observed regarding B cell subsets between the DM and healthy control groups. Breg cells could predict DN (AUC = 0.720, p = 0.017) at a cut-off of ≤13.5% with 95% sensitivity and 95% specificity. In conclusion, Breg cells serve a vital role in suppressing aberrant immune reactivity, meaning that their insufficiency can trigger heightened autoimmune processes. Ultimately, the Breg subpopulation holds substantial promise as a significant biomarker for predicting both the onset and progression of DN with no correlation with the severity of DN.