In the current study, we aimed to clone the full-length cDNA of porcine CRTC3 (pCRTC3) gene and examine its expression pattern and function in intestinal epithelial cells. The full-length cDNA sequence of pCRTC3 was 2,173 bp (GenBank accession no. MF964215), with a 1,860-bp open reading frame encoding a 620-AA protein. Comparison of the deduced AA sequence with different species including human, mouse, rat, Papio, cattle, and rabbit showed 89% to 91.9% similarity. The pCRTC3 was highly expressed in small intestine and spleen, to a lesser degree in lung, liver, and adipose tissue, and was expressed at a low but detectable level in skeletal muscle, kidney, and heart. In addition, high protein levels of pCRTC3 were found in IPEC-J2 cells, in which pCRTC3 was mainly localized in cytoplasm. Furthermore, we demonstrated that knockdown of pCRTC3 significantly decreased the expression of the porcine tight junction-related genes including zonula occludens-1 (ZO-1), ZO-2, occludin, and claudin-1 by 57.88% (P < 0.01), 40.19% (P < 0.01), 51.59% (P < 0.01), and 35.70% (P < 0.05), respectively. Taken together, we first cloned the full-length sequence of pCRTC3 and revealed the tissue-specific expression pattern, localization, and function of pCRTC3 in regulating the expression of intestinal tight junction-related genes. This study could provide some useful information for understanding the function of CRTC3 in pigs.