Objective To construct specific small interfering RNA (siRNA) expression vectors of transketolase-like gene 1 and to detect the silencing effects. Methods A plasmid pEGFP-C1-U6/TKTL1 containing human U6 snRNA promoter was ligated to a 19 bp reverse repeated motif of TKTL1 target sequence with 9 bp spacer. The recombinant pEGFP-C1-U6/TKTL1 plasmids were identified by restriction endonuclease (Pst I and Sal I) and DNA sequencing, and then transferred into LoVo cell line. The level of TKTL1 mRNA was examined by real-time PCR. Results The TKTL1 short hairpin RNA (shRNA) expression frame was successfully inserted into expression vector. Enzyme digestion by Pst I and Sal I showed a 400 bp DNA fragment. The sequence of shRNA recombinant plasmid was the same as that of the designed fragment. Conclusions Expression vectors of shRNA specific for TKTL1 were constructed. The expression of TKTL1 mRNA in LoVo cell line transfected by recombinant pEGFP-C1-U6/TKTL1 plasmids was reduced significantly.