The multifunctional Y-Box protein 1 (YB-1) exerts positive and negative regulatory effects on gene expression by different mechanisms. Since transcription can be controlled by micro RNAs (miRNAs), YB-1 could also cause effects on gene expression by regulation of cellular miRNAs. To test this hypothesis, a previously established and well-characterized cell model derived from drug-sensitive (EPG85-257P/tetR/YB-1) and multidrug-resistant (EPG85-257RDB/tetR/YB-1) gastric carcinoma cells, in which the expression of YB-1 can be inhibited by tetracycline-dependent triggering of the RNA interference (RNAi) pathway, was investigated concerning their miRNA expression profiles in the presence and absence of YB-1. Microarray hybridizations demonstrated that six miRNAs (miR-96*, miR-210, miR-503, miR-623, miR-1275, miR-1290) were up-regulated more than 1.5-fold in drug-sensitive cells following YB-1 inhibition, but no differences in miRNA expression could be detected in multidrug-resistant cells. Independent validation of these findings by quantitative real-time reverse transcriptase polymerase chain reaction did not confirm these effects. Likewise, an in silico analysis of potential regulatory effects of the miRNAs on their target genes did not support the potential miRNA regulatory effects of YB-1. In conclusion, the data provide evidence that YB-1 has no direct influence on global miRNA expression pattern in different variants of gastric carcinoma cells and, therewith, does not control gene expression by regulation of miRNAs.