α-Linolenic acid (ALA) serves as a precursor of long-chain ω-3 fatty acids, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Humans cannot produce ALA due to a lack of ω-3 (Δ-15) fatty acid desaturase (FAD), which converts linoleic acid (LA) into ALA in plants. We developed ALA hyperfortified intragenic rice via a double T-DNA transformation strategy, with an expression cassette containing endogenous ω-3/Δ15 fatty acid desaturase (FAD3) gene, endosperm-specific GluC promoter, and GluB-5 terminator in one T-DNA, and the selectable marker Hpt gene in the other. The Hpt-containing T-DNA was removed from the self-pollinated progeny. The ALA content of the intragenic rice seeds increased up to 6.44 mg/g, representing a 14.93-fold increase over the wild-type control. The ALA, EPA, and DHA concentrations in the brain and serum of rats fed with the ALA-enriched intragenic rice increased significantly. The ALA-enriched rice could provide a sustainable and safe solution to combat ω-3 fatty acid deficiency.