Rho GDP-dissociation inhibitor β (RhoGDIβ), a regulator of the Rho family of proteins, is expressed abundantly in the hematopoietic cell lineage. During apoptosis of hematopoietic cells, RhoGDIβ is cleaved by caspase‑3 at Asp19 and this cleaved form (Δ19‑RhoGDIβ) has been implicated in the apoptotic pathway. To clarify the role of RhoGDIβ in hematopoietic cells, the present study performed immunoblotting and immunofluorescence staining to examine the expression of RhoGDIβ and ∆19‑RhoGDIβ during phorbol 12‑myristate 13‑acetate (PMA)‑stimulated differentiation of human THP‑1 monocytic cells to macrophages. During differentiation of the THP‑1 cells to macrophages, the expression of RhoGDIβ remained stable; however, the expression of Δ19‑RhoGDIβ increased, particularly in well‑spreading, non‑apoptotic cells, which differentiated into macrophages. These results suggested that Δ19‑RhoGDIβ has an apoptosis‑independent role in the PMA‑induced differentiation of THP‑1 cells to macrophages.