The development of deep sequencing has enabled the identification of novel microRNAs (miRNAs), leading to a growing appreciation for the fact that individual miRNAs can be heterogeneous in length and/or sequence. MiRNA variants carrying heterogeneous 3’ ends, termed 3’ isomiRs, are the most frequently observed type of isomiR, in terms of both the number of miRNAs displaying these variations and their overall abundance1 . Through deep-sequencing of Argonaute-associated small RNAs, we identified widespread 3’ modifications of miRNAs. Using miR-222 isoforms as the model, we showed that, despite having the identical seed sequence, 3’ isomiRs could impart drastically different phenotypic outcomes and possessed differential targeting effects. Whilst growth arrest was observed for the canonical miR-222, extensive cell death was induced specifically by its 3’ isomiRs. It is clear from deep sequencing data that the sequences of many miRNAs vary from the standard sequences reported in miRBase. This has potentially dramatic implications for the miRNA researcher, because the reagents used to measure and manipulate the levels of miRNA may be designed against a different sequence from that predominantly expressed in vivo. Therefore, we caution researchers to take 3’ isomiRs into consideration in their experiments.