Plants require adequate water for growth, development, and reproduction. Peptides play a key role in plant growth and development and act in a similar manner to plant hormones. However, only a few peptides have been identified to play a role in abiotic stress tolerance in potato. In this study, we identified fourteen members of the epidermal patterning factor (EPF) family in potato, which were designated as StEPF1-14 according to their chromosomal locations. We also conducted a comprehensive analysis of their chromosomal distribution, gene structures, physicochemical properties, phylogenetic relationships, and tissue-specific expression patterns. RT-qPCR analysis revealed that the StEPF4 gene is significantly induced by drought stress, suggesting its potential role as a negative regulator in the plant’s response to drought. Furthermore, multiple cis-regulatory elements associated with drought-responsive regulation were identified within the promoter region of the StEPF genes. Here, we isolated an EPF secreted Cys-rich small peptide StEPF4 from ‘Atlantic’ and explored its mechanism in plant response to drought stress. We found that StEPF4 was greatly induced by dehydration treatment in potato. To investigate its potential biological functions, StEPF4 was knocked down in potato. The StEPF4 knocked down lines (KdStEPF4) significantly decreased stomatal density, resulting in a decrease in the transpiration rate. KdStEPF4 lines maintained a higher photosynthetic rate and lowered the water loss rate of leaves compared with the control, resulting in increased drought resistance. Taken together, this study provides detailed information about StEPFs, and our findings also show that StEPF4 plays an essential role in regulating drought resistance by reducing stomatal density in potato.