The maize (Zea mays ssp. mays) wild ancestor, teosinte, has three times the seed protein content of most modern inbreds and hybrids, but the mechanisms responsible for this trait are unknown. We created a contiguous haplotype DNA sequence of a teosinte, Zea mays ssp. Parviglumis, with Trio-Binning, and map-based cloned a major high-protein QTL, teosinte high protein 9 (Thp9) on chromosome 9. Thp9 encodes an asparagine synthetase 4 that is highly expressed in teosinte, but not in the B73 inbred, where a deletion in the 10 th intron of Thp9-B73 causes incorrect splicing of Thp9-B73 transcripts. Transgenic expression of Thp9-teosinte in B73 significantly increased seed protein content. Introgression of Thp9-teosinte into modern maize inbreds and hybrids greatly enhanced free amino acid accumulation, especially asparagine, throughout the plant, increasing seed protein content without affecting yield. Thp9-teosinte appears to increase nitrogen utilization efficiency, important for promoting a high yield under low nitrogen conditions.