Verticillium dahliae is a soil-borne pathogenic fungus that causes Verticillium wilt in host plants, a particularly serious problem in potato cultivation. Several pathogenicity-related proteins play important roles in the host infection process, hence, identifying such proteins, especially those with unknown functions, will surely aid in understanding the mechanism responsible for the pathogenesis of the fungus. Here, tandem mass tag (TMT) was used to quantitatively analyze the differentially expressed proteins in V. dahliae during the infection of the susceptible potato cultivar “Favorita”. Potato seedlings were infected with V. dahliae and incubated for 36 h, after which 181 proteins were found to be significantly upregulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that most of these proteins were involved in early growth and cell wall degradation. The hypothetical, secretory protein with an unknown function, VDAG_07742, was significantly upregulated during infection. The functional analysis with knockout and complementation mutants revealed that the associated gene was not involved in mycelial growth, conidial production, or germination; however, the penetration ability and pathogenicity of VDAG_07742 deletion mutants were significantly reduced. Therefore, our results strongly indicate that VDAG_07742 is essential in the early stage of potato infection by V. dahliae.