Gene knockout is an effective approach to investigate gene function of high-molecular-weight glutenin subunit (HMW-GS). In this study, we developed a set of single HMW-GS deletion lines of Ningmai 9 (soft wheat) to understand the effects of HMW-GS deletion on kernel quality, quantity of gluten protein fractions and processing quality. Among the 29 lines tested, three lines were wild type and the remaining were knockout mutants including Glu-A1x, Glu-B1x,Glu-B1y, Glu-D1x, and Glu-D1y deletion types of five, seven, five, five, and four lines, respectively. Compared with the wild type, the HMW-GS deletion lines had no significant difference in flour protein content, wet gluten content, kernel hardness, and solvent retention capacity. All the single HMW-GS deletion types decreased the ratios of glutenin-to-gliadin and HMW-GS-to-LMW-GS in quantity, and the decreases in Glu-B1x and Glu-D1x deletion types were significant at P < 0.05. In the wild type, mixograph peak time was 2.00 min and TIMEX width was 4.57%; whereas, those in the HMW-GS deletion lines were significantly lower (P < 0.05), varying from 1.38 min to 1.64 min and from 3.38% to 3.98%, respectively. Particularly, the Glu-B1x and Glu-D1x deletion lines showed the lowest mixograph peak time and TIMEX width. Although similar sugar snap cookie diameter was observed among the five deletion types, Glu-B1x, Glu-B1y, and Glu-D1y deletion types showed significantly higher cookie diameter than the wild type (P < 0.05). In this study, single deletion of HMW-GS weakened gluten strength and improved sugar snap cookie processing quality, indicating that HMW-GS knockout can be used to improve soft wheat quality.