Tumorigenesis requires energy production via aerobic glycolysis (Warburg effect) in malignant tumors. Recent research has demonstrated that the pentose phosphate pathway (PPP) is augmented in some tumors, especially the non-oxidative part of the PPP which is controlled by transketolase (TKT) enzyme reactions. One TKT isoform, transketolase-like protein 1 (TKTL1), is specifically upregulated in different human cancers, and its overexpression predicts poor patient survival. To further define the function of in malignant progression, we employed the small interference RNA (siRNA) technique to knockdown gene expression of TKTL1 in the gastric cancer cell line AGS. We used TKTL1 siRNA to observe the effect of reduced TKTL1 expression on gastric cancer tumorigenesis in a nude mice xenograft model and on proliferation in vitro. Our results showed that the expression of double stranded RNA led to the efficient and specific inhibition of endogenous TKTL1 expression in AGS cells. In addition, the TKT activity was significantly deceased in the TKTL1 siRNA-treated AGS cells. TKTL1 suppression resulted in delayed cell proliferation in vitro. Furthermore, loss of TKTL1 inhibited the growth of AGS tumor xenografts. Altogether, our findings indicate that the specific inhibition of TKTL1 may be important therapeutically.