To explore the role of the miRNA-1297/phospholipase C beta 1 (PLC beta 1) axis in intestinal barrier injury. Abnormally expressed miR-1297 and its target gene PLC beta 1 as well as their transcriptome sequencing were confirmed by bioinformatics analysis. Next, the intestinal barrier injury was induced by lipopolysaccharide (LPS) in the CCCHIE-2 cells. Subsequently, the impacts of miR-1297 and PLC beta 1 on the transcriptome were estimated. QRT-PCR and Western blotting were conducted to detect the relative mRNA and protein expressions, respectively. The cell viability and permeability were analyzed by MTT assay and fluorescent yellow detection. miR-1297 was significantly upregulated in patients with human immunodeficiency virus/acquired immunodeficiency syndrome and targeted PLC beta 1. Moreover, overexpressed PLC beta 1 was mainly enriched in the transforming growth factor-beta signaling pathway, while the knockdown of miR-1297 was focused on the arginine biosynthesis pathway. The overexpression of miR-1297 could reduce the PLC beta 1 expression and inhibit the viability of CCCHIE-2 cells injured by LPS, while the effect of the downregulation of miR-1297 was on the opposite. Western blotting and cell fluorescence localization experiments revealed that the inhibition of miR-1297 increased the expressions of PLC beta 1 and ZO-1. In addition, the upregulation of miR-1297 strengthened the permeability in cells injured by LPS, as did the knockdown of PLC beta 1. miR-1297 could restrain the repair of intestinal barrier injury via negatively regulating PLC beta 1 and its tight junction downstream protein ZO-1 in CCC-HIE-2 cells injured by LPS, which indicated that PLC beta 1 and miR-1297 might be important targets for the repair of intestinal barrier injury.