Virus-encoding RNA-dependent RNA polymerase (RdRp) is essential for genome replication and gene transcription of human coronaviruses (HCoVs), including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We previously identified the interaction between the catalytic subunit NSP12 of SARS-CoV-2 RdRp and the host protein CREB-regulated transcription coactivator 3 (CRTC3), a member of the CRTC family that regulates cyclic AMP response element-binding protein (CREB)-mediated transcriptional activation. Currently, the implication of CRTC3 in the pathogenesis of HCoVs is poorly understood. Herein, we demonstrated that CRTC3 attenuates RdRp activity and SARS-CoV-2 genome replication, therefore reducing the production of progeny viruses. The interaction of CRTC3 with NSP12 contributes to its inhibitory effect on RdRp activity. Furthermore, we expanded the suppressive effects of two other CRTC family members (CRTC1 and CRTC2) on the RdRp activities of lethal HCoVs, including SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV), along with the CREB antagonization. Overall, our research suggests that CRTCs restrict the replication of HCoVs and are antagonized by CREB, which not only provides new insights into the replication regulation of HCoVs, but also offers important information for the development of anti-HCoV interventions.Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.