Gallbladder cancer (GBC), the most common malignancy of the biliary tract, shows late diagnosis and low survival rate and requires continued search for new diagnostic biomarkers and therapeutic targets. Human endogenous retroviruses (HERVs) are specifically prone to be reactivated in diverse cancers and are implicated in cancer progression and immunotherapy.Single-cell RNA sequencing was performed on tumor tissues and paired adjacent tissues from 4 GBC patients. Dual-luciferase reporter assay was applied to measure enhancer activity of HERV sequences.We dissected the cellular diversity and described the HERV transcriptomic landscape for GBC. We found that HERVs were transcribed in a cell type-specific manner and different HERV families were associated with diverse biological effects. HERVs could function as enhancers, presumably causing altered expression of neighboring genes. The transcription level of HERVH was gradually elevated with the malignant transformation of epithelial cells, suggesting HERVH may be a potential early diagnostic biomarker of GBC. HHLA2, a newly emerging immune checkpoint, was derived by HERVH, exhibited an expressional correlation with HERVH, and was identified as a promising target for immunotherapy.Exploring the transcriptional landscape and potential functional impact of HERVs highlights the important role of HERVs in GBC and provides a fresh perspective on managing GBC.This study was supported by the National Natural Science Foundation of China (31970176, 81972256) and the research grants from the Innovation Capacity Building Project of Jiangsu province (BM2020019).