The topoisomerase II inhibitor idarubicin (Ida) is an effective anticancer anthracycline drug and has been used for clinical therapies of multiple cancers. It is well-known that Ida and its analogues can induce DNA double strand breakage (DSB) by inhibiting topoisomer II and kill tumor cells. To date, it remains unknown whether they alter DNA epigenomes. Here, we show that Ida significantly stimulates the oxidation of a key epigenetic mark DNA 5-methyl-2'-deoxycytidine (5mC), which results in elevation of 5-hydroxymethyl-2'-deoxycytidine (5hmC) in four tested cell lines. Similarly, Ida analogues also display elevated 5hmC. DSB-causing topoisomer II inhibitor etopside fails to induce 5hmC change even at very high dose, which suggests the independence of the DSB. Moreover, the structure comparison supports that the histone eviction-associated amino sugar moiety is a characteristic of the anthracyclines required to promote the 5hmC elevation. Noteworthy, we also found that the 5mC oxidation is also cell-cycle dependent and mainly occurs during the S and G2/M phases. TET2 depletion diminishes the observed 5hmC elevation, which suggests that the Ida stimulation of 5hmC formation is mainly TET2-dependent. Deep-sequencing shows that 5hmC increases in all regions of the tested genome of T47D cells. The observation of a novel effect of Ida as well as other anthracycline compounds on epigenetic DNA modifications may help to further elucidate their biological and clinical effects.