Verticillium wilt caused byVerticillium dahliaeis a notorious soil-borne fungal disease and seriously threatens the yield of economic crops worldwide. During host infection,V. dahliaesecretes many effectors that manipulate host immunity, among which small cysteine-rich proteins (SCPs) play an important role. However, the exact roles of many SCPs fromV. dahliaeare unknown and varied. In this study, we show that the small cysteine-rich protein VdSCP23 inhibits cell necrosis inNicotiana benthamianaleaves, as well as the reactive oxygen species (ROS) burst, electrolyte leakage and the expression of defense-related genes. VdSCP23 is mainly localized in the plant cell plasma membrane and nucleus, but its inhibition of immune responses was independent of its nuclear localization. Site-directed mutagenesis and peptide truncation showed that the inhibition function of VdSCP23 was independent of cysteine residues but was dependent on theN-glycosylation sites and the integrity of VdSCP23 protein structure. Deletion ofVdSCP23did not affect the growth and development of mycelia or conidial production inV. dahliae. Unexpectedly,VdSCP23deletion strains still maintained their virulence forN. benthamiana,Gossypium hirsutumandArabidopsis thalianaseedlings. This study demonstrates an important role for VdSCP23 in the inhibition of plant immune responses; however, it is not required for normal growth or virulence inV. dahliae.