Abstract: Nowadays, application of miRNases—artificial ribonucleases aimed at degradation of noncoding RNAs, in particular, miRNAs—represents one of the novel experimental approaches to inhibit tumorigenesis. miRNases integrate in their structure an addressing oligonucleotide, which provides specific binding with miRNA target, and a catalytic group, which promotes cleavage of the RNA substrate. Introduction of chemical modifications to the oligonucleotide domain of miRNases in the region that is complementary to miRNA may significantly increase the hybridization properties and nuclease resistance of this type of compound. However, the influence of such structural changes to the ribonuclease activity of miRNases remains unclear. In this work, to investigate the effect of 2'OMe modifications on the activity of miRNases, we synthesized two types of anti-miRNA-21 conjugates of the peptide [(ArgLeu)2Gly]2 and hairpin oligonucleotides in which 14-mer binding region to the miRNA target was fully or partially modified. It is shown that the introduction of 2'OMe modifications promotes a considerable increase in the affinity of miRNases to miRNA-21 but does not change significantly their nuclease resistance. Full modification of conjugates in the region that is complementary to miRNA negatively affects their ribonuclease activity, whereas partial introduction of 2'OMe nucleotides considerably enhances the cleavage activity of miRNases, which leads to a substantial decrease in the proliferation rate and migration potential of tumor cells, which are determined by the miRNA-21 expression. Keywords: oligonucleotide–peptide conjugates, oncogenic miRNA, miRNA-21, 2'OMe modification, human epidermoid carcinoma KB-8-5.