Heterogeneous nuclear ribonucleoprotein L – hnRNP L is an RNA binding protein master regulator of alternative splicing, mRNA stabilization, transcription and translation. hnRNP L enhances alternative splicing of various target genes. With regard to muscle diseases, hnRNP L deficiency results in impaired myogenesis in human primary muscle cells and abnormal hnRNP L foci develop in myoblasts from humans affected by myotonic dystrophy type 1 (DM1). The Notch signaling pathway is essential for regulating muscle satellite cell quiescence and myogenesis, and the deficiencies of the Notch pathway genes (MEGF10, JAG2 and POGLUT1) have been implicated with muscular dystrophies. We used shRNA techniques to knock down endogenous hnRNP L expression in C2C12 cells and the Gal4-UAS system to knock down smooth (the Drosophila ortholog of hnRNP L) in fruit flies. We found that hnRNP L knockdown in C2C12 cells altered myotube formation during differentiation and subsequently led to downregulation of the differentiation markers MyoD and MyoG and specific Notch pathway target genes, including Notch1, Hes1 and Hey1. In C2C12 cells, hnRNP L knockdown led to differential expression during specific stages of myogenesis for the following Notch pathway genes: MEGF10, JAG2 and POGLUT1. In Drosophila, myogenesis stage specific knockdown of Smooth using Gal4 drivers showed that smooth/hnRNP L plays an essential role in early differentiation and mature muscle fiber formation coinciding with decreased Notch and Twist (embryonic/adult muscle precursor) activities. In murine Muscle stem cells, siRNA mediated hnRNP L knockdown resulted in downregulation of several Notch pathway genes including JAG2.This provides additional evidence that hnRNP L might play a vital role in modulating muscle development and disease mechanisms in part by influencing the gene expression patterns of multiple Notch pathway components. Hence hnRNP L might be explored further as a common therapeutic target for these rare muscle diseases involving Notch signaling pathway dysfunction.