Tendon cells exist in a dense extracellular matrix and mechanical loading is important for the strength development of this matrix. We therefore use a three‐dimensional (3D) culture system for tendon formation in vitro. The objectives of this study were to elucidate the temporal expression of tendon‐related genes during the formation of artificial tendons in vitro and to investigate if early growth response‐1 (EGR1), EGR2, FOS, and cyclooxygenase‐1 and ‐2 (PTGS1 and PTGS2) are sensitive to mechanical loading. First, we studied messenger RNA (mRNA) levels of several tendon‐related genes during formation of tendon constructs. Second, we studied the mRNA levels of, for example, EGR1 and EGR2 after different degrees of loading;​ dynamic physiologic‐range loading (2.5% strain), dynamic overloading (approximately 10% strain), or tension release. The gene expression for tendon‐related genes (i.e., EGR2, MKX, TNMD, COL3A1) increased with time after seeding into this 3D model. EGR1, EGR2, FOS, PTGS1, and PTGS2 did not respond to physiologic‐range loading. But overloading (and tension release) lead to elevated levels of EGR1 and EGR2 (p ≤ 0.006). FOS and PTGS2 were increased after overloading (both p < 0.007) but not after tension release (p = 0.06 and 0.08). In conclusion, the expression of tendon‐related genes increases during the formation of artificial tendons in vitro, including EGR2. Furthermore, the gene expression of EGR1 and EGR2 in human tendon cells appear to be sensitive to overloading and unloading but did not respond to the single episode of physiologic‐range loading. These findings could be helpful for the understanding of tendon tensional homeostasis.