This study aims to investigate the bioaccessibility, bioactivity and gut microbiota modulation effect of Moringa oleifera Lam. leaves after in vitro gastrointestinal digestion and colonic fermentation. A total of 24.95 ± 0.91 mg GAE per g DM and 21.66 ± 1.41 mg RE per g DM of phenolics and flavonoids were released during digestion. The HPLC-ESI-TOF/MS analysis showed that the major phenolic compound 6,8-di-C-glucosylapigenin was released during oral digestion, catechin during gastric digestion, and quercetin-3-O-β-d-glucoside and ferulic acid during small intestine digestion. The antioxidant activity of Moringa oleifera Lam. leaves during the whole digestion process was in line with the release of phenolics and flavonoids. In addition, the fermentation of Moringa oleifera Lam. leaves induced the production of acetic, propionic, and n-butyric acids leading to a decrease in pH, as well as the growth of beneficial colonic bacteria. The results suggested that Moringa oleifera Lam. leaves are a promising candidate for healthy food.