Background: Shift work, especially during nighttime hours, disrupts the circadian system and is linked to higher rates of metabolic, gastrointestinal, cardiovascular, and neurocognitive disorders. Emerging evidence suggests that gut microbiota may mediate these associations. This systematic review assessed whether shift work alters gut microbiota composition and explored potential health consequences. Methods: A systematic search was conducted in PubMed, Scopus, and ScienceDirect from inception to March 2025. Studies reporting gut microbiota alterations in adult shift workers were included. Two reviewers independently screened articles and extracted data. Risk of bias was assessed using the NIH Quality Assessment Tool and the ROBINS-E framework. Five studies met the eligibility criteria and were included in the final synthesis. Results: The selected studies comprised four observational investigations with small sample sizes and one Mendelian randomization study leveraging large-scale genetic datasets. Observational studies reported reduced α-diversity and increased relative abundance of pro-inflammatory genera—including Escherichia/Shigella, Blautia, and Dialister—in night shift workers. These microbiota alterations were associated with gastrointestinal complaints and indicators of cardiometabolic dysfunction. The Mendelian randomization study provided preliminary evidence supporting a causal relationship between circadian misalignment, gut dysbiosis, and increased cardiovascular risk. Conclusions: Shift work is associated with significant alterations in gut microbiota composition that may contribute to adverse health outcomes. However, current evidence is limited and heterogeneous, preventing firm causal conclusions. Further high-quality longitudinal and interventional research is needed to clarify underlying mechanisms and inform preventive strategies.