Introduction: Spider silks are the strongest and most elastic fibers known in the nature. Cotton is also one of the most important crops in the world which increasing the quality of its fibers is of great importance in the textile industry, so the production of longer and stronger cotton fibers is of great importance. The aim of this study was to produce transgenic cotton fibers containing MaSp1 cobweb protein. Material and Methods: A synthetic construct was designed based on a selected fragment of Latrodectushesperus MaSp1 gene. The gene was then cloned twice inpCAMBIA1304 binary vector using different restriction enzymes (NcoI and NheI; EcoR1 and NheI). Agrobacterium tumefaciens LBA4404strain was transformed by recombinant vectors which are used to transform cotton plants. The accuracy of the results was confirmed using PCR, hygromycin resistance gene and recombinant protein expression. Finally, the fiber strength of transgenic cotton was measured by two vectors. Result: MaSp1 synthetic gene was transferred to the plant meristem. The study of transgenic plants in the next generations confirmed the accuracy of gene transfer and expression of recombinant protein. Conclusion: Considering the economic role of cotton in the world and also having the third rank among the industrial plants of the country, this study succeeded in producing transgenic cotton plants expressing Masp1 protein with increased fiber quality.