This study investigates the effects of space microgravity on human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and the potential role of thiamine in mitigating these effects. During spaceflight, hPSC-CMs exposed to microgravity exhibited reduced beating rate, abnormal intracellular calcium cycling, and metabolic remodeling, particularly in thiamine metabolism. Thiamine, a vitamin B1 essential for energy metabolism, was found to be blocked in hPSC-CMs under microgravity conditions, leading to decreased tricarboxylic acid (TCA) cycle efficiency and ATP production. This resulted in cytoskeletal remodeling and calcium homeostasis imbalance. In vitro and in vivo studies showed that thiamine supplementation could reverse these adaptive changes induced by simulated microgravity. The study provides insights into the mechanisms of microgravity-induced cardiac adaptations and suggests that thiamine supplementation may be a feasible countermeasure against microgravity-induced cardiac dysfunction during long-duration spaceflight.This study investigates the effects of space microgravity on human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and the potential role of thiamine in mitigating these effects. During spaceflight, hPSC-CMs exposed to microgravity exhibited reduced beating rate, abnormal intracellular calcium cycling, and metabolic remodeling, particularly in thiamine metabolism. Thiamine, a vitamin B1 essential for energy metabolism, was found to be blocked in hPSC-CMs under microgravity conditions, leading to decreased tricarboxylic acid (TCA) cycle efficiency and ATP production. This resulted in cytoskeletal remodeling and calcium homeostasis imbalance. In vitro and in vivo studies showed that thiamine supplementation could reverse these adaptive changes induced by simulated microgravity. The study provides insights into the mechanisms of microgravity-induced cardiac adaptations and suggests that thiamine supplementation may be a feasible countermeasure against microgravity-induced cardiac dysfunction during long-duration spaceflight.