Efforts to understand the impact of spaceflight on the human body stem from growing interest in long-term space travel. Multiple organ systems are affected by microgravity and radiation, including the cardiovascular system. Previous transcriptomic studies have sought to reveal the changes in gene expression after spaceflight. However, little is known about the impact of long-term spaceflight on the mouse heart in vivo. This study focuses on the transcriptomic changes in the hearts of female C57BL/6J mice flown on the International Space Station (ISS) for 30 days. RNA was isolated from the hearts of three flight and three comparable ground control mice and RNA sequencing was performed. Our analyses showed that 1147 transcripts were significantly regulated after spaceflight. The MAPK, PI3K-Akt, and GPCR signaling pathways were predicted to be activated. Transcripts related to cytoskeleton breakdown and organization were upregulated, but no significant change in the expression of extracellular matrix (ECM) components or oxidative stress pathway-associated transcripts occurred. Our results indicate an absence of cellular senescence, and a significant upregulation of transcripts associated with the cell cycle. Transcripts related to cellular maintenance and survival were most affected by spaceflight, suggesting that cardiovascular transcriptome initiates an adaptive response to long-term spaceflight.