The position of the nucleus in a cell is controlled by interactions between the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the cytoskeleton[Crisp, 2006]. Defects in nuclear positioning are often associated with muscle weakness and dysfunction, suggesting that proper nuclear localization and anchorage is essential for normal skeletal muscle function[Gundersen 2013]. Nesprin 1, which includes multiple isoforms, is an integral component of the LINC complex critical for nuclear positioning and anchorage in skeletal muscle, and is thought to provide an essential link between nuclei and actin [Zhang 2010]. Importantly, Nesprin 1G and Nesprin 1α2 are the predominant isoforms of Nesprin 1 expressed in skeletal muscle[Randles 2010]. We, and others, have previously shown that Nesprin 1 is critical for nuclear positioning and anchorage in skeletal muscle[Zhang 2010;Puckelwartz 2009;Zhang 2007]. Notably, loss of all known Nesprin 1 isoforms led to postnatal lethality in 60% of newborn pups, and surviving mice developed skeletal myopathy[Zhang, 2010]. Nesprins are thought to regulate nuclear anchorage by providing a critical link between nuclei and the actin cytoskeleton[Zhang 2010;Puckelwartz, 2009;Zhang, 2007] therefore previous approaches to study the role of Nesprin 1 in skeletal muscle either interfere with the KASH domain[Puckelwartz 2009;Zhang 2007] or ablate all Nesprin 1 isoforms[Zhang 2010]. However, there is currently no direct evidence to suggest Nesprin 1G links the nucleoskeleton to actin filaments in skeletal muscle, and current studies preclude the understanding as to which isoform of Nesprin 1 is critical for skeletal muscle function. To address this question, and to investigate the in vivo function of different Nesprin 1 isoforms, we generated actin binding-deficient Nesprin 1 (Nesprin 1ΔCH-/-) mice, in which the exon encoding the actin-binding region of Nesprin 1 was ablated, and Nesprin 1α2 isoform-specific deficient mice (Nesprin 1α2-/-). Using a combination of immunofluorescence, biochemistry and functional assays, we show that actin binding is dispensable for postnatal viability, nuclear positioning, and skeletal muscle function. In contrast, loss of Nesprin 1α2 led to severe nuclear mispositioning, and postnatal lethality. Furthermore, the few surviving Nesprin 1α2 KO developed kyphosis, indicative of skeletal muscle dysfunction. Interestingly, we found that the microtubule motor protein, Kinesin 1, was specifically mislocalized in Nesprin 1α2-/- muscle fibers, but remained at the NE in skeletal muscles of Nesprin 1ΔCH-/- mice. These data suggest that Nesprin 1α2 plays a fundamental role in vivo and is the critical Nesprin 1 isoform essential for skeletal muscle function. Furthermore, Nesprin 1α2 interacts with Kinesin 1 to facilitate the nuclear dynamics necessary to position nuclei for normal skeletal muscle function.