Setosphaeria turcica (syn. Exserohilum turcicum) is the pathogenic fungus of maize (Zea mays) that causes northern leaf blight, which is a major maize disease worldwide. Melanized appressoria are highly specialized infection structures formed by germinated conidia of S. turcica that infect maize leaves. The appressorium penetrates the plant cuticle by generating turgor, and glycerol is known to be the main source of the turgor. Here, the infection position penetrated by the appressorium on maize leaves was investigated, most of the germinated conidia entered the leaf interior by directly penetrating the epidermal cells, and the appressorium structure was necessary for the infection, whether it occurred through epidermal cells or stomata. Then, to investigate the effects of key factors in the development of the appressorium, we studied the effects of three inhibitors, including a melanin inhibitor (tricyclazole, TCZ), a DNA replication inhibitor (hydroxyurea, HU), and an autophagy inhibitor (3-methyladenine, 3-MA), on appressorium turgor and glycerol content. As results, appressorium turgor pressure and glycerol concentration in the appressorium reached their highest levels at the mature stage of the appressorium under the control and inhibitor treatments. The three inhibitors had the greatest effects on appressorium turgor pressure at this stage. Glycogen and liposomes are the main substances producing glycerol. It was also found inhibitors affected the distribution of glycogen and liposomes, which were detected in the conidia, the germ tube, and the appressorium during appressorium development. This study provides profound insight into the relationship between appressorium turgor pressure and glycerol content, which was affected by the synthesis of melanin, DNA replication, and autophagy in the developing appressorium during a S. turcica infection.