Objective To construct a lentivirus vector for RNA interference targeting myosin Va gene and to observe its effect on motility and migration of human pulmonary giant cell carcinoma PG cells. Methods Based on the efficient target sequence for myosin Va RNAi, two pairs of oligo DNA containing myosin Va RNAi target sequence or scramble sequence were synthesized and inserted into pSuper vector, followed by sequence analysis. The expressing cassette H1 promoter-shM5A/shCON from the recombinant pSuper plasmid was then transferred to the lentivirus vector plenti4, and the recombinant lentivirus was packaged. PG cells were transduced with the packaged lentivirus and the positive cells were screened by zeocin selection. RT-PCR was performed to determine the myosin Va RNAi efficiency in zeocin-resistant PG cells, and wounding assay and Boyden chamber assay were utilized to examine the capabilities of motility and migration in myosin Va RNAi PG cells. Results Restriction enzyme digestion and sequencing confirmed the successful construction of the lentivirus vector containing myosin Va RNAi target or scramble sequence. RT-PCR result showed that myosin Va mRNA levels were remarkably reduced in lentivirus-based myosin Va RNAi PG cells. The abilities of motility and migration were also significantly inhibited in lentivirus-based myosin Va RNAi PG cells, as demonstrated in wounding assay and Boyden chamber assay.Conclusion Myosin Va RNAi lentivirus vector was successfully constructed and efficiently repressed myosin Va expression in PG cells. Repression of myosin Va by RNAi led to the inhibition of PG cells motility and migration, indicating that there might exist correlation between the expression of myosin Va and cancer progression.