Although influenza DNA vaccine research has focused mainly on viral hemagglutinin and has led to promising results, other virion proteins have also shown some protective potential. In this work, we explored the potential of a DNA vaccine based on the PB1 protein to protect BALB/c mice against lethal influenza A virus infection. The DNA vaccine consisted of pTriEx4 plasmid expressing PB1. As a positive control, a pTriEx4 plasmid expressing influenza A virus HA was used. Two weeks after three subcutaneous doses of DNA vaccine, the mice were challenged intranasally with 1 LD50 of A/Puerto Rico/8/34 (H1N1) virus, and PB1- and HA-specific antibodies, survival rate, body weight change, viral mRNA load, infectious virus titer in the lungs, cytokines IL-2, IL-4 and IL-10, and granzyme-B were measured. The results showed that (i) the PB1-expressing DNA vaccine provided a fair protective immunity in the mouse model and (ii) viral structural proteins such as PB1 represent promising antigens for DNA vaccination against influenza A.