The diversity and complexity of the cancer transcriptome may contain transcripts unique to the tumor environment. Here, we report a LIN28B variant, LIN28B-TST, which is specifically expressed in hepatocellular carcinoma (HCC) and many other cancer types. Expression of LIN28B-TST is associated with significantly poor prognosis in HCC patients. LIN28B-TST initiates from a de novo alternative transcription initiation site that harbors a strong promoter regulated by NFYA but not c-Myc. Demethylation of the LIN28B-TST promoter might be a prerequisite for its transcription and transcriptional regulation. LIN28B-TST encodes a protein isoform with additional N-terminal amino acids and is critical for cancer cell proliferation and tumorigenesis. Our findings reveal a mechanism of LIN28B activation in cancer and the potential utility of LIN28B-TST for clinical purposes. Display omitted •RNA-seq analyses reveal a LIN28B tumor-specific variant in cancers•LIN28B-TST initiates from an ATI site regulated by NFYA but not c-Myc•LIN28B-TST expression is controlled by DNA methylation•LIN28B-TST is critical for cancer cell proliferation and tumorigenesis Guo et al. identified a tumor-specific LIN28B transcript variant, LIN28B-TST, in hepatocellular carcinoma and many other cancer types produced by alternative transcription initiation. The LIN28B-TST-expressing tumors may represent a subtype of aggressive cancer. LIN28B-TST could serve as an ideal and promising target candidate for cancer diagnosis and therapy.