Tubulin tyrosine ligase like 12 (TTLL12), a member of the tubulin tyrosine ligase (TTLL) family, has not been completely characterized to date. It is reported that histone methylation, tubulin modifications, mitotic duration and chromosome ploidy play crucial roles in a variety of cancers, and are related to tumorigenesis and cancer progression. A recent study showed that TTLL12 may be a pseudo-enzyme which has a SET-like domain and a TTL-like domain. In the present study, we first used 3'-rapid amplification of cDNA ends (3'-RACE) to amplify the transcripts of the TTLL12 gene from a human lung cancer cell line H1299, and unexpectedly discovered a new transcript isoform characterized with an additional 108-bp nucleotide sequence inserted at the location from 902 to 903 bases of the TTLL12 coding sequence (CDS), where it also locates between exons 5 and 6. Next, utilizing RT-PCR and Sanger sequencing, we further confirmed the existence of such a new transcript isoform of TTLL12 in more human cancer cells including lung cancer cells and other cancer cells. Moreover, several lung cancer cell lines were found to display a much higher proportion of the new isoform compared with TTLL12 wild-type transcript. These results suggest that the new TTLL12 isoform may be of importance for proper maintenance of lung cancer cells. Therefore, the new isoform of TTLL12, with the inserted sequences probably acting as a disordered region, provides a novel perspective regarding TTLL12 functions in human cancers including lung cancer.