The human genome encodes two proteins closely related to transketolase (TKT), transketolase-like protein 1 and 2 (TKTL1 and TKTL2). TKTL1 shares 61% amino acid sequence identity with TKT. More and more evidence suggests a role for TKTL1 in proliferation or cell cycle regulation. Indeed, TKTL1 is overexpressed in various cancers and is correlated with poor prognosis in colon, urothelial, gastric, and lung cancers as well as in ocular adnexa carcinomas, rectum carcinomas, and laryngeal squamous cell carcinomas. Increased TKTL1 levels also correlate with esophageal squamous cell carcinoma metastasis and increased resistance against cisplatin chemotherapy in nasopharyngeal carcinomas. Moreover, TKTL1 overexpression promotes cell proliferation and enhanced tumor growth; in contrast, TKTL1 down regulation attenuates the proliferation of various types of cancer cells. However, the cellular functions and regulatory machineries of TKTL1 are still largely unknown. Through this proteomic analysis, we defined the protein-protein interaction map for the TKTL1 and uncovered the novel regulating function of TKTL1 in regulating TKT transketolase activity.