Plant cell suspension cultures are composed of a homogenous population of cells to get a stress signal in a similar manner, even at short-term microgravity. In this study, the effect of suborbital flight was investigated on the growth mechanisms of Matricaria chamomilla cells for the first time by studying cell viability, signaling molecules, and the antioxidant defense system. For the flight experiment, cells were cultured in Petri dishes; placed in the bio-capsule during suborbital flight (4 min microgravity), and harvested for several analyses after 5 and 10 days of flight. Results showed that suborbital flight decreased cell growth, which was related to a decline in cell viability. Cell morphology is changed from elongated to a round shape at the stationary growth phase, which was higher in flight samples comparing to the control. Hydrogen peroxide (H2O2) and Ca2+ levels increased significantly under suborbital flight, especially after 5 days of flight (P ≤ 0.05). Peroxidase activity showed a considerable rise after 5 and 10 days of flight by 68.07% and 28.96% comparing to the control, respectively. Moreover, flavonoid content reached the maximum level (1.55 mg g-1 FW) in cells exposed to suborbital flight after 10 days comparing to the control (1.01 mg g-1 FW). Findings of this study showed that signaling molecules of H2O2 and Ca2+ play an important role in regulating cell growth and viability after flight by inducing peroxidases.