MicroRNAs (miRNAs) regulate gene expression post-transcriptionally by fine-tuning mRNA levels and translation. miRNA biogenesis is tightly regulated to maintain specific miRNA expression patterns in different tissues and developmental stages, misexpression leading to pathological conditions. miRNAs are transcribed as longer precursors that undergo multiple processing steps before the mature miRNAs reach their target mRNAs in the cytoplasm. Although the RNA binding proteins (RBPs) Drosha, DGCR8 and Dicer are the essential components of the miRNA processing pathway, other RBPs, have recently been identified as critical regulators of miRNA biogenesis. The CNNC motif bound by the SR protein splicing factor SRSF3 has been shown to specify pri-miRNA hairpins and in vitro studies have demonstrated SRSF3 enhancing pri-miRNA processing in a CNNC dependant manner. However, the in vivo functional relevance of SRSF3 activity in miRNA processing has remained unexplored. Our analysis of SRSF3 binding sites in embryonic stem cells (ESCs) identified SRSF3 binding at the CNNC motif particularly in miRNAs located in polycistronic miRNA clusters. We demonstrate that SRSF3 depletion in ESCs leads to reduced levels of mature miRNAs without affecting the levels of the pri-miRNAs. Intriguingly, the processing of specific miRNAs within miRNA clusters is individually regulated, leading to differential expression of mature miRNAs derived from the same pri-miRNA. Furthermore, SRSF3 is frequently overexpressed in tumour cells and misexpression of miRNAs is a characteristic of many cancers. Our analysis of SRSF3, pri-miRNA, miRNA and miRNA-target expression in human tumour-normal pairs demonstrates that SRSF3 may confer some of its pathological properties through the control of miRNAs. Thus, our work reveals a novel mechanism regulating the hallmark properties of cancer cells and highlights the roles of multifunctional RBPs in gene regulation in health and disease.