Natural killer (NK) cells play an important role in preventing cancer development. NK group 2 member D (NKG2D) is an activating receptor expressed in the membrane of NK cells. Tumour cells expressing NKG2DL become susceptible to an immune-dependent rejection mainly mediated by NK cells. The paradoxical roles of transforming growth factor beta (TGF-β) in regulation of NKG2DL are presented in many studies, but the mechanism is unclear. In this study, we showed that TGF-β up-regulated the expression of NKG2DLs in both PC3 and HepG2 cells. The up-regulation of NKG2DLs was characterized by increasing the expression of UL16-binding proteins (ULBPs) 1 and 2. TGF-β treatment also increased the expression of transcription factor SP1. Knockdown of SP1 significantly attenuated TGF-β-induced up-regulation of NKG2DLs in PC3 and HepG2 cells, suggesting that SP1 plays a key role in TGF-β-induced up-regulation of NKG2DLs. TGF-β treatment rapidly increased SP1 protein expression while not mRNA level. It might be due to that TGF-β can elevate SP1 stability by activating PI3K/AKT signalling pathway, subsequently inhibiting GSK-3β activity and decreasing the association between SP1 and GSK-3β. Knockdown of GSK-3β further verified our findings. Taken together, these results revealed that AKT/GSK-3β-mediated stabilization of SP1 is required for TGF-β induced up-regulation of NKG2DLs. Our study provided valuable evidence for exploring the tumour immune modulation function of TGF-β.