Recently, several efforts in space exploration have been devoted to understand its effect on the human body, also to meet the objective of NASA to send humans to an asteroid by 2025 and to Mars in the 2030. Spaceflight conditions impose numerous ambient alterations, including the reduction of gravity (microgravity, µG) and increased solar radiation, leading to adverse health effects. Thus, the investigation of the potential changes and stressors that astronauts may experience during spaceflight is becoming crucial. Due to the high costs and limitations of experiments in space, on-ground methods simulating µG represent a suitable alternative to preliminary investigate µG effects on cell models. In this work human colon adenocarcinoma Caco-2 cells were selected as model of intestinal epithelial barrier and their response to simulated µG was investigated with label-free shotgun proteomics and bioluminescent reporter gene assays. The Rotary Cell Culture System-1 bioreactor, developed by NASA, was modified with 3D-printed adaptors to hold T25 culture flasks. A cell-based bioluminescence assay to assess the transcriptional regulation in normal gravity and μG of NF-κB promoter, involved in inflammation signalling. NanolucP luciferase, the destabilized version of Nanoluc luciferase, was selected as reporter because, thanks to its reduced half-life, its expression closely reflects changes in transcriptional activity and provides an increased signal-to-background ratio. Caco-2 cells were grown for 72 h in G or μG and transiently transfected with a reporter plasmid carrying NanoLucP under the control of NFκB-RE. Basal transcription of NFkB and its responsiveness to external stimuli (incubation for 5 h at 37°C with concentration range from 0.1 to 10 ng mL−1 TNF-α) were evaluated. Exposure to μG reduces both NFκB basal activation (8.4 ± 0.6-fold reduction) and induction by TNFα (1.2 ± 0.1-fold reduction, in the presence of 10 ng mL-1), corroborating the hypothesis of reduced immunity in microgravity conditions.