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Chronic pain is a debilitating threat to human health, and its molecular mechanism remains undefined. Previous studies have illustrated a key role of cAMP response element-binding protein (CREB) in pain regulation; CREB-regulated transcription coactivator 1 (CRTC1) and microRNA212/132 (miR212/132) are also vital in synaptic plasticity. However, little is known about the interaction among these factors in pain condition. We conducted this experiment mainly to determine the crosstalk between CREB, CRTC1, and miR212/132 in vitro. Moreover, we explored the changes in hyperalgesia on chronic constrictive injury (CCI) mouse in vivo when given CREB-related adenovirus vectors, CRTC1-related adenovirus vectors, and miR212/132-locked nucleic acid (LNA).We cultured primary neurons in the spinal cord of mouse embryos. Exogenous glutamate was added to cultured neurons to simulate in vivo pain process. Real-time quantitative polymerase chain reaction was used to determine changes of NR2B, CRTC1, CREB, and miR212/132 at the mRNA level; Western blot was used to detect p-NR2B, p-CREB, and CRTC1 at protein level. Von Frey cilia were used to study mechanical hyperalgesia in a murine model of CCI. CREB-miR (adenovirus vector interfering CREB gene), CREB-AD (adenovirus vector overexpressing CREB gene); CRTC1-miR (adenovirus vector interfering CRTC1 gene), CRTC1-AD (adenovirus vector overexpressing CRTC1 gene), and miR212/132-LNA were injected intrathecally.In vitro, 100 μmol/L glutamate induced p-CREB and miR212/132-LNA. CRTC1 protein was downregulated by CREB-miR and miR212/132-LNA. CRTC1 mRNA was upregulated by CREB-AD and downregulated by CREB-miR and miR212-LNA. P-CREB was upregulated by CRTC1-AD and downregulated by miR212/132. CREB mRNA was upregulated by CRTC1-AD and downregulated by CRTC1-miR. MiR212/132 was upregulated by CRTC1-AD and CREB-AD; downregulated by CREB-miR. In vivo, CRTC1-miR, CREB-miR, and miR212/132-LNA increased paw withdrawal mechanical threshold in various degrees.The NR2B-CREB-miR212/132-CRTC1-CREB signal network plays an important role in the regulation of pain. Intervening with any molecule in this signal network would reduce pain perception.