Late flowering in lettuce produces additional leaves and maintains the edible quality of its product. Control of flowering time in plants is vital for plant fitness and for agricultural purposes and is an important factor in successful reproduction and seed crop yield and depends on the environmental and internal parameters of the plant and it is controlled by the regulatory network of genes. One of the problems in the field of crops and horticulture is the mismatch of flowering time with favorable environmental conditions. by increase in population, it is necessary to produce efficient plants with higher biomass. In the flowering regulatory network, Flowering Locus C (FLC), a MADS box transcription factor, prevents flowering. The autonomous pathway accelerates the onset of flowering independently of daylight by suppressing FLC. The MSI4/FVE gene is a key gene in the autonomous pathway. It is involved in controlling cell proliferation and early differentiation, organ growth and plays a key role in accelerating flowering by reducing the expression of FLC gene via epigenetic changes. in this study, using the RNA Silencing technique, MSI4/FVE gene expression was repressed at post transcriptional level. To this end, first, the tissue culture of lettuce in MS culture medium was optimized using BAP, Kinetin and NAA hormones for direct regeneration from cotyledon and leaf explants.  The MS medium containing 0.1 mg/l NAA and 0.01 mg/l BAP has the highest regeneration rate for cotyledon explants. Then, for construction of silencing hairpin construct, a fragment of Arabidopsis thaliana MSI4 gene was amplified by PCR and then cloned in sense and in antisense directions into pFGC5941 vector by two consecutive cloning. The hairpin structure in this recombinant vector was then transferred to the lettuce plant by Agrobacterium. The obtained transgenic plants were tested by PCR to confirm the presence of the transgene and the study on the flowering phenotype of these transgenic plants showed that the silencing of MSI4 in lettuce caused the plant to flower late (about 1.5 months delay) and produce additional leaves.