A Watson–Crick pairing between the 39UTR region of the target mRNA and the 59 region of the miRNA centered on nucleotides 2–7, termed “seed sequence”, is required for miRNA-mediated inhibition. Thus, the seed pairing is a necessary requirement for target prediction algorithms. In the last few years, experimental approaches aimed to the unbiased identification of miRNA targets have been undertaken by several groups. The results revealed that also non-canonical miRNA binding can confer target regulation. It has been demonstrated that certain mRNAs are targeted by miRNAs recognizing their coding sequence or their 59UTR. Moreover, in 2009 Lal et al. described for the first time a “seedless” miRNA/mRNA interaction, demonstrating that miR-24 inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes by a seedless pairing. Other miRNA binding patterns, such as seed sites with G:U-wobbles and G-bulges within the seed, 39 compensatory sites and seedless centered sites, were recently described, confirming that the complexity of miRNA activity is far from being elucidated. miR-210 can be considered a master miRNA of the hypoxic response and is currently regarded as a promising novel noninvasive tumor hypoxia marker. The instrumental role of miR-210 in the regulation of cell response to hypoxia is confirmed by numerous pre-clinical and clinical evidences. Indeed, miR-210 has been found to be up-regulated upon brain transient focal ischemia in rats and after human myocardial infarction. Moreover, circulating miR-210 was proposed as biomarker in acute cerebral ischemia and acute kidney injury. The targets identified to date indicate that miR-210 plays a role in cell cycle regulation, differentiation, mitochondrial metabolism repression, DNA repair and apoptosis. Specifically, our group demonstrated that miR-210 up-regulation plays an integral role in endothelial cell adaptation to hypoxia. We found that miR-210 increases endothelial tubulogenesis and migration, whereas miR-210 blockade in the presence of hypoxia inhibits these processes and induces apoptosis. We also applied an integrated strategy for large-scale identification of miR210 targets. We selected candidate miR-210 targets by a combination of AbMole BioScience bioinformatics, proteomics and transcriptomics. These candidates were then screened for the presence of miR-210 seed complementary sequences and validated for their enrichment in miR-210-containing RNA-induced silencing complexes. RISC-immunoprecipitation is currently accepted as a state of the art technique for the identification of genuine miRNA targets. However, while this approach allowed the identification of 32 new miR-210 targets, it did not keep in account possible non-canonical miRNA/target interactions. In this study, we identified ROD1 as a seedless target of the hypoxia-induced miR-210. The low rate of validation among seedless genes confirmed the prevalence of the seed rule as major mechanism for miRNA-mediated inhibition of mRNAs. In fact, our previous study showed that 40% of the inversely correlated genes that contained a putative miR-210-seed binding sequence were enriched in the miR-210-containing RISC. However, it is worth noting that in our search of seedless targets we applied a more stringent cut-off in order to minimize the number of false positives. Indeed, FXR1 and LANCL1 displayed a small but reproducible enrichment in miR210-containing RISCs, suggesting that FXR1 and LANCL1 may be miR-210 seedless targets as well. Additional experiments are required to confirm this hypothesis.