The second mitochondria‑derived activator of caspases (Smac/DIABLO), is a mitochondrial protein that is released along with cytochrome c during apoptosis and promotes caspase activity. It has been reported that c‑Jun N‑terminal kinase (JNK) is involved in the regulation of Smac release during apoptosis; however, the specific role of JNK is not well understood. The aim of the present study was to investigate whether JNK1 is activated during apoptosis induced by tumor necrosis factor‑α (TNF‑α) and whether the activation of JNK1 is functionally associated with Smac release from the mitochondria in HeLa cells. It was determined that during apoptotic progression induced by TNF‑α, JNK1 is activated and translocated into the mitochondria. It was also shown that Smac release is markedly promoted by transient expression of JNK1, while it is suppressed by the expression of a dominant negative version of JNK1. Furthermore, expression of JNK1 also increases the levels of TNF‑α‑induced caspase‑3 activity. To determine whether JNK1 activity is directly involved in the release of Smac, an in vitro phosphorylation assay was performed with the N‑terminal 10‑mer peptide fragment of Smac (pep0110). The results indicated that pep0110 is phosphorylated in dose and time‑dependent manners by active JNK1, and that the phosphorylation of Smac at the N‑terminal serine 6 residue is functionally linked to Smac release during TNF‑α‑induced apoptosis. In conclusion, this suggests that Smac is a major physiological substrate of JNK1 in the regulation of apoptosis.