X-box binding protein 1 (XBP1), a vital basic leucine zipper transcription factor for the related gene transcription in endoplasmic reticulum (ER) stress, belongs to the CREB/ATF family. In mammals, XBP1S is the activated one of XBP1 isoform. In order to study the role of fish XBP1S, we cloned and identified the XBP1S (KU509247) from grass carp (Ctenopharyngodon idella) (named CiXBP1S) by homologous cloning and RACE technique. The full length of CiXBP1S is 1694 bp along with 124 bp of 5′ UTR, 418 bp of 3′ UTR and the longest open reading frame (1152 bp) encoding a polypeptide of 383 amino acids with a well conserved DNA binding domain (BRLZ domain). CiXBP1S shares significant homology to zebrafish XBP1S (˜90%) at amino acid level. RT-PCR showed that the expression of CiXBP1S was ubiquitous in all tested grass carp tissues and was significantly up-regulated under the stimulation with tunicamycin (Tm) in CIK (C. idellus kidney) cells. To study the molecular mechanism of transcriptional regulation for XBP1 signaling pathway in fish, we cloned grass carp XBP1 promoter sequence. Its promoter is 1036 bp in length and divided into two distinct regions in which an ER stress response element (ERSE) exists in the proximal region. Meanwhile, grass carp ATF6 (CiATF6N) and CiXBP1S were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. Gel mobility shift assay showed that CiATF6N and CiXBP1S had the high affinity with CiXBP1 promoter sequence in vitro. Co-transfection of pcDNA3.1-CiATF6 (or pcDNA3.1-CiXBP1S respectively) with pGL3-CiXBP1P2 (or pGL3-CiXBP1P1 respectively) into epithelioma papulosum cyprini (EPC) cells showed that CiATF6 and CiXBP1S played a positive role in CiXBP1S transcription. CiXBP1S also had high affinity with CiGRP78 and CiGRP94 promoter sequences. In addition, recombinant plasmids of pGL3-CiGRP78P and pGL3-CiGRP94P were constructed and transiently co-transfected with pcDNA3.1-CiXBP1S (pcDN3.1-CiXBP1S-nBRLZ, respectively) into EPC cells. The result showed that CiXBP1S can activate CiGRP78 and CiGRP94 promoters.