Neuronal cell death-inducible putative kinase (NIPK) is a protein with an unknown function encoded by a gene activated in neuronal cells in cell death-causing conditions (disruption of calcium homeostasis, trophic factor deprivation). Using the yeast two-hybrid screening of an embryonic mouse cDNA library, we identified activating transcription factor 4 (ATF4) as a protein binding to mouse (m) NIPK. The critical domain for mNIPK-binding resides in a 72 amino acid stretch near the N-terminus of ATF4, covering the second leucine zipper motif and the preceding region. mNIPK expressed as fusion protein with enhanced yellow fluorescence protein (EYFP) is localized predominantly in the nucleus, and the mNIPK-ATF4 complex can be immunoprecipitated from cells cotransfected with epitope-tagged mNIPK and ATF4 constructs. The expression of both mNIPK and ATF4 is upregulated in the neuronal cell line GT1-7 in response to disruption of calcium homeostasis by thapsigargin, but ATF4 is induced more rapidly than mNIPK. The coexpression of mNIPK inhibits ATF4 CRE-dependent transcriptional activation activity in transiently transfected cells. At the same time, ATF4 degradation rate is not increased in the cells coexpressing mNIPK, and ATF4, associated to mNIPK, is able to bind to CRE. Thus, mNIPK is a novel regulator of ATF4 transcriptional activity.