Abstract: Objective To investigate the mechanism of tRF-1:32-Glu-TTC-1 in mediating high glucose (HG)-induced pyroptosis in renal tubular epithelial cells (RTECs). Methods RTECs were treated with high glucose (35 mmol/L), and the expression of tRF-1:32-Glu-TTC-1 was detected by RT-PCR. The morphological changes of these cells were observed by scanning electron microscopy (SEM). The mRNA levels of NLRP3 and GSDMD were detected by RT-PCR, and the protein levels of NLRP3, caspase-1, and GSDMD-N were measured by Western blot. RTECs were transfected with tRF-1:32-Glu-TTC-1 inhibitor, and the transfection efficiency was detected by RT-PCR. The mRNA and protein levels of pyroptosis-related indicators were detected by Western blot. The protein levels of p-PI3K/PI3K, p-Akt/Akt, and p-FoxO3a/FoxO3a were measured by Western blot. Results Compared with the Control group, the HG group showed increased expression of tRF-1:32-Glu-TTC-1, and pyroptosis was observed by SEM. The mRNA and protein levels of pyroptosis-related indicators was upregulated. The tRF-1:32-Glu-TTC-1 inhibition group showed lower mRNA and protein levels of pyroptosis-related indicators than the HG group and the inhibitor blank vector group. Compared with the Control group, decreased levels of p-PI3K/PI3K, p-Akt/Akt, and p-FoxO3a/FoxO3a proteins were observed in the HG group. The tRF-1:32-Glu-TTC-1 inhibition group presented higher protein levels of p-PI3K/PI3K, p-Akt/Akt, and p-FoxO3a/FoxO3a than the HG group and the inhibitor blank vector group. Conclusions tRF-1:32-Glu-TTC-1 may promote HG-induced pyroptosis in RTECs through negatively regulating the PI3K/Akt/FoxO3a signaling pathway.