Objective To construct and identify rat myosin heavy chain 14 (MYH14) gene recombined lentiviral vector by RNA interference technique. Methods Based on the MYH14 mRNA sequence, a single-stranded primer was designed to form a double-stranded oligonucleotide sequence, which was ligated into the GV298 lentiviral vector linearized by AgeⅠ and BamHⅠ double enzymes restriction, and then the bacterial liquid was verified by PCR and sequencing, respectively. The plasmid was extracted in the bacterial liquid with correct sequence and transfected into rat Schwann cells RSC96. The transfection efficiency was observed by immunofluorescence, the shRNA plasmid could effectively knock down MYH14 was screened by Western blotting, and the cell viability of RSC96 cells after transfection was detected by CCK-8. Results Three pairs of MYH14-shRNA sequences were synthesized and cloned into GV298 vector to construct recombinant plasmids MYH14-shRNA1, 2, and 3, and the vector MYH14-shRNA1 and MYH14-shRNA2 were screened by PCR and sequencing. Immunofluorescence showed that the cell fluorescence was the strongest at 72 h after transfection. Western blotting analysis showed that compared with the negative control (scramble sequence) group, the expression level of MYH14 protein in RSC96 cells was significantly decreased after MYH14-shRNA2 transfection (0.57±0.15 vs 1.11±0.06, P<0.01), while there was no significant difference after MYH14-shRNA1 transfection (P>0.05). There was no significant difference in cell viability of RSC96 cells between the negative control and MYH14-shRNA2 groups 24 h after transfection (1.09±0.16 vs 1.00±0.15, P>0.05). Conclusion The rat MYH14 gene recombinant lentiviral vector has been successfully constructed, which can effectively down-regulate the expression of MYH14 in RSC96 cells.