Increasing evidence suggest that the glucose-lowering drug metformin exerts a valuable anti-senescence role. The ability of metformin to affect the biogenesis of selected microRNAs (miRNAs) was recently suggested. MicroRNA isoforms (isomiRs) are distinct variations of miRNA sequences, harboring addition or deletion of one or more nucleotides at the 5' and/or 3' ends of the canonical miRNA sequence. We performed a comprehensive analysis of miRNA and isomiR profile in human endothelial cells undergoing replicative senescence in presence of metformin. Metformin treatment was associated with the differential expression of 27 miRNAs (including miR-100-5p, -125b-5p, -654-3p, -217 and -216a-3p/5p). IsomiR analysis revealed that almost 40% of the total miRNA pool was composed by non-canonical sequences. Metformin significantly affects the relative abundance of 133 isomiRs, including the non-canonical forms of the aforementioned miRNAs. Pathway enrichment analysis suggested that pathways associated with proliferation and nutrient sensing are modulated by metformin-regulated miRNAs and that some of the regulated isomiRs (e.g. the 5' miR-217 isomiR) are endowed with alternative seed sequences and share less than half of the predicted targets with the canonical form. Our results show that metformin reshapes the senescence-associated miRNA/isomiR patterns of endothelial cells, thus expanding our insight into the cell senescence molecular machinery.