Through extensive studies of plant immunity, many positive regulators including PBS3 and EDS1 and negative regulators including NPR3 and NPR4 have been identified. The function of each individual immune regulator has been well characterized. However, the relationships among these important immune regulators remain elusive. Event though it is well known that EDS1 plays essential roles in plant basal defense, effector-triggered immunity, and salicylic acid-mediated plant immunity, more work will be needed to gain a better understanding on how EDS1 is regulated. Here, we show that PBS3 physically associates with EDS1 in both the cytoplasm and the nucleus. Interestingly, we found that EDS1 protein level is significantly reduced in pbs3 mutants. NPR3 and NPR4, which function as SA receptors and adaptors for Cullin3-based E3 ligase, interact with and mediate the degradation of EDS1 via the 26S proteasome. We proved that PBS3 inhibits the poly-ubiquitination and subsequent degradation of EDS1 by reducing the association of EDS1 with NPR3 and NPR4. Our study revealed a unique mechanism, in which plants fine-tune defense responses by inhibiting the degradation of a positive player in plant immunity.