Objective To design, construct and select the optimal repl ication-defective recombinant adenovirus mediated short hairpin RNA (shRNA) which is transduced into human osteosarcoma cells to silence c-myc gene expression, and to construct the recombinant adenovirus vector expressing c-myc-shRNA and determine its viral titer. Methods Three pairs of complementary single-stranded ol igonucleotides (ss ol igos) were designed and synthesized, and then they were annealed to create a double-stranded ol igonucleotide (ds ol igos).The ds ol igos were cloned into pENTR/U6 vector to produce the shuttle plasmid pENTR/U6-shRNA, which was transduced into osteosarcoma cells by l iposome after sequencing. The plasmid with good silence effect was chosen by RT-PCR to perform the LR recombination reaction to the adenovirus backbone plasmid. The expression clone was transfected into HEK293A cells to produce repl ication-incompetent recombinant adenovirus mediated shRNA against c-myc whose cytopathic effect was observed and viral titer was determined by the viral particle (VP) method and 50% tissue culture infective dose (TCID50). Results Ds ol igos, which was verified by electrophoresis, was cloned into pENTR/U6 vector to produce pENTR/U6-shRNA shuttle plasmid, which was confirmed to be corrected by sequencing. The optimal plasmid with good silence effect was chosen by RT-PCR from the three pairs of double-stranded ol igonucleotide. By Pac I enzyme, the l inearrization repl ication-defective recombinant adenovirus mediated shRNA was constructed to perform the LR recombination reaction to the adenovirus backbone plasmid. The cytopathic effect and vacuole phenomenon of adenovirus mediated shRNA appeared at 3 days and became obvious at 6 days. The adenovirus virus titer in the first generation was 5.23 × 109 VP/mL, and reached 2.26 × 1012 VP/mL via 3-4 generations’ ampl ification. The viral titer was 10-3.8/0.1 mL determined by VP method and TCID50. Conclusion The recombinant adenovirus mediated shRNA c-myc is constructed in vitro through RNA interference technology.