DNA damage repair pathways greatly affect the response to genotoxic drugs in cancer cells, so inhibition of such pathways could be a potentially useful strategy to enhance chemosensitivity. DNA-dependent protein kinase (DNA-PK) plays a crucial role in the repair of DNA double-strand breaks (DSBs) that are probably one of the most detrimental types of DNA damage. It has been shown that DNA-PK is highly expressed in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. Less well appreciated was the effect of DNA-PK inhibition on sensitivity of BCP-ALL cells to DNA-damaging agents. Here, we show that the DNA-PK inhibitor NU7441 increased doxorubicin-induced apoptosis in BCP-ALL cell lines (NALM-6, SUP-B15), correlating with a reduction in DSB repair measured by γ-H2AX foci. NU7441 affected the cell cycle distribution and the cell cycle regulatory molecules in combination with doxorubicin treatment. Doxorubicin-induced DNA-PK phosphorylation was decreased in the presence of NU7441. Apoptosis induction by the combined treatment was associated with marked reduction of Bcl-2 and survivin and a significant increase of Bax mRNA expression levels. In conclusion, our data indicate that inhibition of DNA-PK might be an effective approach to enhance the tumor-cell-killing effects of DNA-damaging agents such as doxorubicin in BCP-ALL and may deliver novel, targeted therapy into the clinic.