NIPBL, a cohesin loader, has been implicated in transcriptional control and genome organization. Mutations in NIPBL, cohesin, and its deacetylase HDAC8 result in Cornelia de Lange syndrome. We report activation of the RNA-sensing kinase PKR in human lymphoblastoid cell lines carrying NIPBL or HDAC8 mutations, but not SMC1A or SMC3 mutations. PKR activation can be triggered by unmodified RNAs. Gene expression profiles in NIPBL-deficient lymphoblastoid cells and mouse embryonic stem cells reveal lower expression of genes involved in RNA processing and modification. NIPBL mutant lymphoblastoid cells show reduced proliferation and protein synthesis with increased apoptosis, all of which are partially reversed by a PKR inhibitor. Non-coding RNAs from an NIPBL mutant line had less m(6)A modification and activated PKR activity in vitro. This study provides insight into the molecular pathology of Cornelia de Lange syndrome by establishing a relationship between NIPBL and HDAC8 mutations and PKR activation.