H7N9 avian influenza virus (AIV) has caused huge losses in the poultry industry and impacted human public health security, and still poses a potential threat. Currently, immune prevention and control of avian influenza relies on traditional inactivated vaccines; however, they have some limitations and genetically engineered avian influenza subunit vaccines may be potential candidate vaccines. In this study, a T169A mutation in the HA protein derived from H7N9 AIV A/Chicken/Guangdong/16876 (H7N9-16876) was generated using the baculovirus expression system (BVES). The results showed that the mutant (HAm) had significantly increased thermostability compared with the wild-type HA protein (HA-WT). Importantly, immunizing chickens with HAm combined with ISA 71VG elicited higher cross-reactive hemagglutination inhibition (HI) antibody responses and cytokine (IFN-γ and IL-4) secretion. After a lethal challenge with heterologous H7N9 AIV, the vaccine conferred chickens with 100% (10/10) clinical protection and effectively inhibited viral shedding, with 90% (9/10) of the chickens showing no virus shedding. The thermostability of HAm may represent an advantage in practical vaccine manufacture and application. In general, the HAm generated in this study represents a promising subunit vaccine candidate for the prevention and control of H7N9 avian influenza.