This study investigated the effects of vacuum pressure and osmotic conditions on the impregnation efficiency, quality retention during refrigeration, and microstructure of fresh-cut Annona squamosa L. treated by vacuum impregnation (Ⅵ). The objective was to determine the optimal processing conditions for maintaining the refrigeration quality and extending the shelf life of fresh-cut A. squamosa L.. The results showed that compared with the untreated group, increasing the vacuum level (10 kPa) significantly enhanced the ascorbic acid enrichment (approximately sevenfold) and impregnation degree, but led to excessive tissue compression. Treatment at 40 kPa exhibited excellent performance in promoting Ca²⁺-pectin cross-linking, improving and maintaining flesh firmness, reducing juice loss, and delaying membrane lipid peroxidation. High-osmotic-pressure treatment, despite maintaining the color to a certain extent, exacerbated cellular dehydration, resulting in increased juice loss, elevated malondialdehyde levels, and severe microstructural collapse. In contrast, isotonic treatment effectively enriched exogenous bioactive compounds and maintained high antioxidant activity and stable soluble solids throughout cold storage. Microstructural observations showed moderate cell shrinkage and well-preserved pore network structures. Comprehensive evaluation showed that vacuum combined with isotonic antioxidant impregnation solution demonstrated significant advantages in enhancing texture, stabilizing color, reducing juice loss, and maintaining cell membrane integrity. This study established and validated a synergistic preservation mechanism of “vacuum-assisted mass transfer enhancement + protection against oxidation + calcium-salt crosslinking for structural stabilization”, and systematically optimized the Ⅵ process parameters of fresh-cut A. squamosa L. based on microstructural characterization.