Background: Clinical practices have demonstrated improvements in prognosis of patients with advanced renal cell carcinoma (RCC) by combination therapies with tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors. However, TKI-resistance is inevitable which hinders further improvement in prognosis. In our previous study, we reported regulating glutamine metabolism could bring about re-sensitivity to Sunitinib (Su) in Su-resistant Renal cell Carcinoma (RCC) cells. However, the mechanism of re-sensitivity and whether it applies to other tyrosine kinase inhibitors (TKIs) has been still unknown. Methods: We established Su and Cabozantinib (Cabo) -resistant cells in 3 RCC cell lines (786-O, Caki-1 and ACHN). We analyzed the activity of glutamine metabolism and VEGF pathway, before and after TKI-resistant、furthermore, conducted antitumor effect in vitro and vivo studies to evaluate re-sensitivity to TKI and relationship between VEGF signaling and regulating glutamine metabolism. Results: In all TKI-resistant cells, the overexpression of glutamine metabolism and VEGF signaling were observed. In vitro and vivo study using Su, the regulating glutaminolysis resulted in 40-74% cell-killing effect in 3 Su-resistant cell lines. Whereas the regulating glutaminolysis also resulted in 35-55% cell-killing effect in 3 Cabo-resistant cell lines. Although the antitumor effect was observed in only 786-O among Su-sensitive cells, it was not observed among Cabo-sensitive cells. Moreover, antitumor effect of regulating glutamine metabolism is more remarkable in vivo. When conducting immunostaining of CD31 to evaluate vascular endothelium, angiogenesis was significantly inhibited by regulating glutamine metabolism.Evaluating VEGF signaling in RT-PCR, VEGFR2 expression and VEGF signaling were downregulated, and PTEN upregulated by suppressing glutamine metabolism. Conclusions: By evaluating glutamate metabolism with VEGF analysis in TKI-resistant RCC cells, we have come to understand a part of the phenomenon of re-sensitivity to TKIs. The inhibition of VEGF signaling and its consequent impact on the tumor microenvironment by regulating glutamine metabolism, are considered to be the mechanisms causing re-sensitivity to TKIs in TKI-resistant cells.We could use TKIs more effectively by regulating glutaminolysis to improve prognosis in advanced RCC patients.