Objective To knockout the murine Txnip gene using microinjection of transcription activator-like effector nuclease (TALEN) mRNAs. Methods TALEN knockout site recognizing Txnip was designed by tools on line, then constructed the vectors and assayed its cleavage activity at cellular level. TALEN mRNA was transcribed in vitro and microinjected into C57BL/6J mouse zygotes. F0 mice were verified at DNA level with BamHI and TXNIP-knockout mice were obtained. Results We designed and constructed TALENs which recognized and cut the first exon of Txnip, and got four TXNIP knockout mice, among which two were frameshift mutation, demonstrating that the TXNIP-knockout mice were generated by TALEN technique. Conclusions Microinjection of in vitro transcribed TALEN mRNAs into murine zygotes is a highly effective and convenient way to develop TXNIP-knockout mouse model.