AMP-activated protein kinase (AMPK) and its upstream kinase LKB1 regulate glucose uptake in skeletal muscle. Brain specific kinase 1 (BRSK1) is an AMPK-related kinase activated by LKB1 via Thr189 phosphorylation and is required for neuronal polarization in mammals.  Three BRSK1 isoforms have been reported in the brain (long, short and S1 form), whereas there have been no studies of BRSK1 expression and function in skeletal muscle.  We used L6 muscle cells and mouse skeletal muscle to study the expression and function of BRSK1 in vitro and in vivo. Of those three BRSK1 isoforms, mRNA and protein analyses showed that L6 cells and skeletal muscles abundantly express the short form BRSK1.  To study the function of short form BRSK1 in muscle, wild type and mutant (Thr189Ala) BRSK1 were over-expressed in L6 myotubes and shRNA was used to knock down BRSK1 (58%).  Wild type BRSK1 over-expression had no effect on basal glucose uptake, but increased insulin-stimulated (30nM, 30min) glucose uptake by 51.0 ± 18.7% vs. empty vector control cells (p<0.05).  BRSK1 knockdown decreased basal glucose uptake by 21.3 ± 5.7% (p<0.05) and insulin-stimulated glucose uptake by 22.8 ± 6.9%, (p<0.05)when compared to scrambled shRNA. To study the role of BRSK1 in vivo, short form Brsk1 was expressed in tibialis anterior (TA) muscles by in vivo gene injection and electroporation. Similar to L6 cells, wild type BRSK1 over-expression increased insulin-stimulated glucose uptake in TA (77.7 ± 12.0%, p<0.05), with no effect of the mutant BRSK1. In summary, short form BRSK1 over-expression increases insulin-stimulated glucose uptake in L6 myotubes and skeletal muscle, and BRSK1 knockdown in L6 cells decreases basal and insulin-stimulated glucose uptake. The short form of BRSK1, an AMPK-related Kinase, is highly expressed in skeletal muscle and may function to regulate insulin action.