This study aimed to explore a role of p21Waf1 in γH2AX foci formation and DNA repair as assessed by a Host-Cell Reactivation Assay in wild-type (p21Waf+/+) and p21Waf1-deficient E1A+Ras-transformed cells. p21Waf1+/+ cells have low γH2AX background compared to p21Waf1−/− cells. The treatment with histone deacetylase inhibitor (HDI) sodium butyrate (NaBut) causes to accumulation of γH2AX in p21Waf+/+ cells with little effect in p21Waf−/− cells. Moreover, NaBut inhibits DNA repair in wt cells but not in p21Waf1−/− cells. This could be explained by the weakening of GADD45 and PCNA proteins binding in NaBut-treated p21Waf1-expressing cells but not in p21Waf1−/− cells. We suggest that in wt-ERas cells NaBut activates both p21Waf1 expression and a release of p21Waf1 from the complexes with E1A that leads to suppression of DNA repair and promotes γH2AX persistency. The absence of p21Waf1 is by itself considered by the cell as stressful factor with formation of γH2AX. But the lack of p21Waf1 interferes with an inhibitory effect of NaBut to inhibit DNA repair and thereby to stop concomitant accumulation of harmful mutations. We conclude that p21Waf1 is directly involved in control of genome integrity and DNA repair acting through modulation of the components of the DNA repair machinery.