Transmembrane and ubiquitin-like domain containing 1 (Tmub1) protein has a negative effect on liver regeneration; however, the mechanisms of Tmub1 expression regulation are currently unknown. CCAAT/enhancer-binding protein β (C/EBPβ) is a key transcription factor associated with interleukin-6 (IL-6) in liver regeneration. The present study aimed to investigate the involvement of C/EBPβ in the IL-6‑induced upregulation of Tmub1. A series of Tmub1 luciferase reporter vectors were constructed to detect the effects of IL‑6 on the transcriptional activity of Tmub1. Small interfering RNA and overexpression vectors were employed to either inhibit or enhance the expression of C/EBPβ, respectively, to determine the effect of C/EBPβ on Tmub1 expression in liver cells. Additionally, chromatin immunoprecipitation experiments were performed to determine whether C/EBPβ bound to the Tmub1 gene, and the sequences associated with the regulation of Tmub1 expression by C/EBPβ were subsequently determined. Several potential binding sites of C/EBPβ were identified in the 5'‑upstream region of the Tmub1 gene. C/EBPβ expression was positively correlated with Tmub1 gene expression in rat liver cells. Furthermore, it was demonstrated that C/EBPβ bound to 5'‑upstream sequences of the Tmub1 gene to enhance the promoter activity. The present study demonstrated that C/EBPβ is a key transcription factor, which can positively regulate the expression of Tmub1 during liver cell proliferation through a possible association with IL-6.