Lipoxygenases (Loxs) are dioxygenases that play an important role in plant growth and defense. Loxs affect flour-processing quality in common wheat. A genome-wide association study (GWAS) identified 306 significant single nucleotide polymorphisms (SNPs) related to Lox activity in Chinese wheat accessions. Among them, a novel Lipoxygenase (Lpx) gene TaLpx-B4 was detected on chromosome 3B in a biparental population. Analysis of EMS-mutagenized wheat lines confirmed the role of TaLpx-B4 in modulating Lox activity. The phylogenetic tree of various plant Lpx genes indicated the predominance of 9-Lpx type in common wheat. Further analysis revealed conserved intron number, exon length, and motif number in the TaLpx gene family. GWAS, linkage mapping, and gene annotation collectively showed that 14 out of 29 annotated TaLpx genes played a critical role in regulating the Lox activity in Chinese wheat. The transgenic wheat grains with the Lpx gene family silenced by RNA interference (RNAi) showed significantly lower Lox activity than the wild type. The TaLpx-RNAi-3 line had a significantly reduced starch content and dough stability and thus possessed relatively superior cookie quality in soft wheat. Further analysis of the transcriptome, lipid component, and other metabolites revealed that the silencing of TaLpx genes significantly increased cookie quality via changes in unsaturated fatty acid content as well as starch, sucrose, and galactose metabolism. This study provides new insight into the role of the TaLpx gene family and valuable information to improve cookie quality.