Background <p>Microgravity alters immune cell function, potentially compromising host defense during spaceflight. Because appropriate immune regulation is also critical in chronic inflammatory and autoimmune conditions, insights from spaceflight biology may have broader implications for human health. Monocyte activation via the p44/42 MAPK pathway is central to inflammatory responses, yet the influence of microgravity on this signaling cascade remains incompletely understood. This study aimed to determine how microgravity affects basal and lipopolysaccharide (LPS)-stimulated ERK1/2 kinases (also known as p44/42 MAP kinases) activity in human monocytes, focusing on signaling state redistribution at both single-cell and population levels.</p> Methods <p>Monocytes were cultured during spaceflight under either normal gravity (1G) or microgravity (µG) and exposed to LPS or control conditions. MAPK activity was quantified and analysed to assess basal signaling, stimulus responsiveness, and variability within the population.</p> Results <p>Basal MAPK activity was significantly elevated in µG compared with 1G monocytes (<i>p</i> = 0.0181). LPS stimulation robustly increased MAPK activity in 1G cells (<i>p</i> = 0.0267) but not in µG (<i>p</i> = 0.6752). Although baseline signaling was higher in µG, LPS responses in µG and 1G were not significantly different (<i>p</i> = 0.7905). Under microgravity, the cell population displayed broader signaling distribution and a larger non-responsive fraction. Although baseline signaling was higher in µG net LPS responsiveness was diminished compared with 1G.</p> Conclusion <p>Microgravity redistributes monocyte signaling states, increasing basal ERK1/2 activity while attenuating rapid stimulus-induced activation and expanding the non-responsive cell fraction. These findings provide new mechanistic insight into how microgravity shapes immune signaling and highlight cellular heterogenety as a critical determinant of immune regulation during spaceflight.</p>