Obesity has become a global health problem. Lifestyle modification and medical treatment only appear to yield short-term weight loss. Roux-en-Y gastric bypass (RYGB) is the most popular bariatric procedure, and it sustains weight reduction and results in the remission of obesity-associated comorbidities for obese individuals. However, patients who undergo this surgery may develop hypoglycemia. To date, the diagnosis is challenging and the prevalence of post-RYGB hypoglycemia (PRH) is unclear. RYGB alters the anatomy of the upper gastrointestinal tract and has a combined effect of caloric intake restriction and nutrient malabsorption. Nevertheless, the physiologic changes after RYGB are complex. Although hyperinsulinemia, incretin effects, dysfunction of β-cells and α-cells, and some other factors have been widely investigated and are reported to be possible mediators of PRH, the pathogenesis is still not completely understood. In light of the important role of the gut microbiome in metabolism, we hypothesized that the gut microbiome might also be a critical link between RYGB and hypoglycemia. In this review, we mainly highlight the current possible factors predisposing individuals to PRH, particularly related to the gut microbiota, which may yield significant insights into the intestinal regulation of glucose metabolic homeostasis and provide novel clues to improve the treatment of type 2 diabetes mellitus.