Significance Netrin-1 is frequently overexpressed in aggressive cancers, acting as the antiapoptotic ligand of the dependence receptors DCC and members of the UNC5H family. Accordingly, netrin-1 inhibition induces apoptosis and tumor growth inhibition. We have previously shown that the transcription factor p53 regulates netrin-1 gene expression in cancer cells treated with conventional chemotherapeutic drugs. We show here that netrin-1 is a target gene of the N-terminal p53 isoform Δ40p53, acting independently of full-length p53 activity, thus promoting cell survival. We show that netrin-1 interference, in presence of Δ40p53, triggers apoptosis in cancer and primary cells and tumor growth inhibition in preclinical in vivo models. We propose that netrin-1 inhibition is a promising therapeutic strategy in tumors expressing high levels of Δ40p53.