The critical 8p22 tumor suppressor deleted in liver cancer 1 (DLC1) is frequently inactivated by aberrant CpG methylation and/or genetic deletion and implicated in tumorigeneses of multiple tumor types. Here, we report the identification and characterization of its new isoform, DLC1 isoform 4 (DLC1-i4). This novel isoform encodes an 1125-aa (amino acid) protein with distinct N-terminus as compared with other known DLC1 isoforms. Similar to other isoforms, DLC1-i4 is expressed ubiquitously in normal tissues and immortalized normal epithelial cells, suggesting a role as a major DLC1 transcript. However, differential expression of the four DLC1 isoforms is found in tumor cell lines: Isoform 1 (longest) and 3 (short thus probably nonfunctional) share a promoter and are silenced in almost all cancer and immortalized cell lines, whereas isoform 2 and 4 utilize different promoters and are frequently downregulated. DLC1-i4 is significantly downregulated in multiple carcinoma cell lines, including 2/4 nasopharyngeal, 8/16 (50%) esophageal, 4/16 (25%) gastric, 6/9 (67%) breast, 3/4 colorectal, 4/4 cervical and 2/8(25%) lung carcinoma cell lines. The functional DLC1-i4 promoter is within a CpG island and is activated by wild-type p53. CpG methylation of the DLC1-i4 promoter is associated with its silencing in tumor cells and was detected in 38-100% of multiple primary tumors. Treatment with 5-aza-2′-deoxycytidine or genetic double knockout of DNMT1 and DNMT3B led to demethylation of the promoter and reactivation of its expression, indicating a predominantly epigenetic mechanism of silencing. Ectopic expression of DLC1-i4 in silenced tumor cells strongly inhibited their growth and colony formation. Thus, we identified a new isoform of DLC1 with tumor suppressive function. The differential expression of various DLC1 isoforms suggests interplay in modulating the complex activities of DLC1 during carcinogenesis.