Mammalian intracellular ribonuclease L (RNase L) is a latent endoribonuclease that functions against viral infections as an apoptosis‐inducing protein, and its activity requires intracellular 5′‐end‐triphosphorylated‐2′,5′ oligoadenylates (2‐5A) as an activator. Previously, we showed that RNase L can be activated in human cancer cell line HT1080 by an RNA polymerase I inhibitor, 1‐(3‐C‐ethynyl‐β‐d‐ribo‐pentofuranosyl)cytosine (3′‐ethynylcytidine; ECyd). In ECyd‐treated cells, knockdown of the RNase L resulted in a marked decrease in c‐jun N‐terminal kinase (JNK) phosphorylation, thereby inhibiting apoptosis. We investigate RNase L binding partners by focused proteomic approach using immunoprecipitation with anti‐RNase L IgG and mass spectrometry. We found that the IQ motif‐containing Ras GTPase‐activating‐like protein 1 (IQGAP1) can associate with RNase L, and that phosphorylation occurs on the IQGAP1. ECyd‐induced JNK phosphorylation and apoptosis were inhibited when IQGAP1 was knocked down with a small interfering RNA. These results raise the interesting possibility that the RNase L–IQGAP1 association may regulate JNK phosphorylation in RNase L‐madiated apoptosis. It is likely IQGAP1 works as a regulator in apoptosis.Structured digital abstract MINT‐7990574: RNaseL (uniprotkb:Q05823) physically interacts (MI:0914) with Kinesin‐like protein KIF23 (uniprotkb:Q02241) and Ras GTPase‐activating‐like protein IQGAP1 (uniprotkb:P46940) by anti bait coimmunoprecipitation (MI:0006) MINT‐7990598: RNaseL (uniprotkb:Q05823) physically interacts (MI:0915) with IQGAP1 (uniprotkb:P46940) by anti bait coimmunoprecipitation (MI:0006)