Vitamin A ensures intestinal homeostasis, impacting acquired immunity and epithelial barrier function; however, its role in innate immunity is mostly unknown. Here, we studied the impact of vitamin A in different dextran sulfate sodium (DSS)-induced colitis animal models. Interestingly, more severe DSS-induced colitis was observed in vitamin A-deficient (VAD) mice than in vitamin A-sufficient (VAS) mice; the same was observed in VAD severe combined immunodeficient mice lacking T/B cells. Remarkably, IL-1β production, LC3B-II expression, and inflammasome activity in the lamina propria were significantly elevated in VAD mice. Electron microscopy revealed numerous swollen mitochondria with severely disrupted cristae. In vitro, non-canonical inflammasome signaling-induced pyroptosis, LC3B-II and p62 expression, and mitochondrial superoxide levels were increased in murine macrophages (RAW 264.7) pretreated with retinoic acid receptor antagonist (Ro41-5253). These findings suggest that vitamin A plays a crucial role in the efficient fusion of autophagosomes with lysosomes in colitis.