Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which has recently been associated with regulating insulin sensitivity in skeletal muscle. After direct phosphorylation by the insulin receptor, FAK regulates skeletal muscle glucose uptake through the reorganisation of the actin cytoskeleton and the subsequent translocation of the glucose transporter-4. Both in vivo and in vitro FAK loss of function studies demonstrate impairments in actin remodelling, glucose uptake and glycogen synthesis. FAK is also present within the microvasculature and may play an important role in insulin-stimulated vasodilation. The purpose of this study was to generate the first visual images of the localisation of FAK within human skeletal muscle and its associated microvasculature using immunofluoresent microscopy. Percutaneous muscle biopsies were taken (with 1% lidocaine as a local anaesthetic) from six lean, healthy active males. 5 µm cryosections were incubated with FAK antibodies for visualisation in muscle fibres and combined with lectin and anti-α-smooth muscle actin to identify capillaries and arterioles. Anti-myosin heavy chain type I was used for fibre type differentiation. Muscle sections were also stained with anti-dihydropyridine receptor (DHPR) for visualisation of t-tubules and investigating colocolisation with FAK. Widefield and confocal immunofluorescence microscopy was used to visualise the localisation and distribution of FAK and image quantification was used to determine fibre type differences in the intensity of FAK immunofluorescence. Within the microvascular endothelium and vascular smooth muscle, FAK appeared in clusters rather than a homogenous distribution. In skeletal muscle, type I skeletal muscle fibres contained 21% more FAK than type II fibres (paired samples t-test; P≤0.001). In both cross-sectional and longitudinal planes, FAK was localised at the sarcolemmal and subsarcolemmal regions of the myofibre. In longitudinal fibres, FAK staining showed uniform striations across the muscle fibre, and co-staining with DHPR suggests FAK colocalises with the t-tubules. The greater FAK content in type I skeletal muscle fibres corresponds with the increased insulin sensitivity of these fibres. This first visualisation of FAK in the human skeletal muscle microvasculature and within the sarcolemmal and t-tubule regions, seems to emphasise FAK’s role in regulating insulin sensitivity at these sites. Immunofluorescence is a valuable method for visualising FAK in skeletal muscle and the associated microvasculature and may provide important insights into the mechanisms leading to insulin resistance in sedentary individuals.