Oenococcus oenican exert its function in hostile wine conditions during the malolactic fermentation process. Therefore, it is an important microbial resource for exploring resistance genes. Hsp20 is an important small heat shock protein fromO. oeni. The conserved consensus motif "A-x-x-x-x-G-x-L" of Hsp20 announced its role as a member of the small heat shock protein family. Thehsp20gene fromO. oeniSD-2a was cloned to create the recombinant plasmid pTriEx-Hsp20. The recombinant plasmid was transformed intoEscherichia coliBL21(DE3) competent cells, and the Hsp20 protein was induced by isopropyl-beta-d-thiogalactoside (IPTG). Thehsp20gene fromO. oeniSD-2a was successfully expressed, and a 20-kDa fusion protein was identified by SDS-PAGE. The purified Hsp20 protein was obtained using Ni-affinity chromatography. Additionally, BL21(DE3)/Hsp20 and BL21(DE3)/Ctrl were treated at high temperatures of 42 and 52 degrees C, at pH values of 2.0-12.0, under oxidative shock with 0.1% (v/v) and 0.2% (v/v) H2O2, and under an osmotic shock of 430 and 860 mM NaCl to compare the effects of heterologous expression of the Hsp20 protein fromO. oeniSD-2a for stress resistance. Notably, Hsp20 overexpression showed enhanced resistance than the control strain did when confronted with different elevated stress conditions. The results demonstrated heterologous expression of thehsp20gene fromO. oeniSD-2a significantly improved the resistance of the hostE. colibacteria against stress conditions.