Abstract: Sterol demethylase inhibitors (DMIs) inhibit the activity of C-14α demethylase, interfere with or block the biosynthesis of ergosterol in cell membrane, cause the accumulation of toxic sterol, affect the structure and function of cell membrane, and thus exert antifungal activities. With the widespread application of DMI fungicides, plant pathogens exhibit arisen resistance to these compounds. Here, we discussed the molecular mechanisms of DMI resistance in agriculture and summarized the main causes of the resistance as follows: Point mutations of CYP51 amino acid residues decrease affinity with fungicides; The insertions of sequence in promoter region lead to overexpression of CYP51 gene; Transcriptional activation or sequence insertion in promoter region promotes the overexpression of efflux pump proteins. This study performed an overview of DMI mode actions and molecular resistance mechanism, which will provide a reference for the structural modification and optimization of anti-bacterial compounds, and strategies for the discovery of new target proteins, promoting the sustainable fungicide innovation and the breakthrough in the resistance issues.