Drought is a major environmental factor limiting grapevine production and quality. However, the roles of miRNAs in different grape varieties at the post-transcriptional level during drought are largely unknown. In this study, we investigated the miRNA abundance in a drought-sensitive grapevine (Vitis vinifera L. cv. Cabernet Sauvignon) (CS) and a drought-tolerant grapevine (Berlandiari Ressiguier No.2 × Rupestris Martin) (110R) in response to drought using Illumina sequencing. A total of 488 known and 892 novel miRNAs belonging to 48 miRNA families were identified in CS and 110-R, respectively. Various accumulation trends of miRNAs observed in the differential drought tolerance of grapevine genotypes were further confirmed using stem-loop qRT-PCR. The target transcripts are predominantly associated with biological processes, cellular components, and molecular functions. Several significantly different drought-responsive miRNAs were examined by β -glucuronidase reporter gene activity driven by miRNA promoters in transgenic lines. The results showed drought-induced downregulation of VvmiR169d and VvmiR398a expression and upregulation of VvmiR156b expression. Moreover, we noted that VvmiR156b overexpression significantly decreased primary root length and increased lateral root numbers in Arabidopsis lines and observed a higher relative growth rate in grape calli under drought treatment. Therefore, genotype-dependent stress-responsive regulation of miRNA expression contributes to the differential drought tolerance of grapevines, and this finding provides mechanistic insight into the vital roles of miRNAs in drought-stress environments in grapevines. • MicroRNAs in 110 R and CS show significantly different responses to drought stress. • Once properly activated, drought-responsive miRNAs can provide dominance in drought. • Drought induced VvmiR169d and VvmiR398a downregulation, and VvmiR156b upregulation. • VvmiR156b overexpressing affects the growth of Arabidopsis root and CS calli. [ABSTRACT FROM AUTHOR]