The Silk Road has long attracted researchers from various fields, sparking their interest in ancient cities and the ancient route that connected civilizations. However, the role of horses and grass in the development of the Silk Road has been relatively understudied. This essay presents a novel perspective, revealing the significance of horses and grass in shaping the growth of nomadic cultures and their profound impact on the formation and development of cities along the Silk Road. Through an examination of Persepolis and Samarkand, this paper analyzes the intricate relationship between horses, grass, and the Silk Road. As a means of transportation, horses' agility and endurance allowed merchants and travelers to traverse vast distances, connecting civilizations and promoting economic and cultural exchanges. The abundant grasslands along the Silk Road corridor shaped the way of life and livelihood of nomadic tribes, providing sustenance for their horses. Nomadic tribes acted as intermediaries, facilitating trade between settled civilizations. This facilitated the establishment of trade routes and the emergence of prosperous urban centers. Therefore, the symbiotic relationship between horses, grass, and nomadic tribes played a crucial role in the development and prosperity of the Silk Road.