Purpose: Protective effect of small heat-shock protein (sHSP) against gamma-radiation, which associated with HSP25-induced cell-cycle delay and Bcl-2 induction. We further extended our studies on the possible role of HSP25 on ionizing radiation-induced cell-cycle regulation. Materials and methods: Flow-cytometric analyses were performed for cell-cycle distribution and Western blotting. Kinase or immunocomplex kinase assay were performed for detection of cell-cycle protein expression or activation. Results: Pronounced arrest of G1, S and G2/M phase was observed by 4Gy radiation and these arrests were augmented by hsp25 overexpression. Inhibition of cyclin-D1, and cyclin-E and induction of p21Waf by radiation, which was more pronounced in hsp25 overexpressed cells than control cells, which is associated with increased binding activity of CDK2. S-phase regulator, cyclin-A and its associated CDK2 and CDC2 kinase activities were also increased by irradiation and hsp25 overexpression attenuated these phenomena. In addition, cyclin-B1 expression and its associated kinase activity, which are responsible for the transition of G2 to M phase, were increased by radiation and hsp25 overexpression also decreased these phenomena. Conclusion: HSP25 augmented radiation-induced cell-cycle arrest (G1, S, and G2/M phase) may be caused by the HSP25-mediated cell-growth delay and is associated with radioresistance.