UL16-binding proteins [ULBPs, also termed as retinoic acid early transcripts (RAET1) molecules] are frequently expressed by malignant transformed cells and stimulate anti-tumor immune responses mediated by NKG2D-positive NK cells, CD8(+) alphabeta T cells and gammadelta T cells in vitro and in vivo. In this study, we identified four novel functional splice variants of ULBPs including ULBP4-I, ULBP4-II, ULBP4-III and RAET1G3 in HepG2 liver carcinoma cells, WISH human amnion cells, Hep-2 larynx carcinoma cells and K562 leukemia cells, respectively, by reverse transcription-PCR and T vector cloning strategy. Analysis of alignments of amino acid sequences of the splice variants illustrated that there were important modifications between splice variants and their individual parental ULBP. All ULBP4 splice variants (ULBP4-I, ULBP4-II and ULBP4-III) were type 1 membrane-spanning molecules and had the ability to bind with human NKG2D receptor in vitro. Ectopic expressions of ULBP4 and ULBP4 splice variants resulted in the enhanced cytotoxic sensitivity of target cells against NK cells, which could be blocked by anti-NKG2D mAb. Moreover, co-culture-free soluble forms of ULBP4 splice variants (their alpha1 + alpha2 ectodomains) and RAET1G3 (soluble splice variant of RAET1G2) with NK cells down-regulated the cell surface expression of NKG2D. Finally, immobilized in a plate-bound form of RAET1G3 stimulated NK cells to secrete IFN-gamma. Taken together, all the identified functional splice variants will help to advance our knowledge regarding the overall functions of ULBP gene family.