MicroRNAs (miRNAs) are a class of endogenous, single-stranded, approximately 21 nt in length, non-coding RNAs which play critical roles in plant biological and metabolic processes. Although numerous miRNAs have been identified in many plant species, miRNAs still remain totally unknown in Ribes nigrum. In the study, two miRNAs (rni-miR5021 and rni-miR5185) were firstly identified in Ribes nigrum using an expression sequence tag (EST)-based comparative genomics approach. Subsequently, transgenic analysis suggested that these putative MIR genes encoding rni-miR5021 and rni-miR5185 were able to generate their corresponding miRNA in vivo. Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis indicated that the expression patterns of the MIR genes varied among blackcurrant tissues, and the two mature miRNAs showed higher accumulation in fruits than other tissues. Finally, 47 targets were predicted for the two miRNAs, which are involved in response to stresses and other plant biological processes. These findings will facilitate future studies on the functions and regulatory mechanisms of miRNAs in Ribes nigrum.