The movement of insulin granules along microtubules, driven by kinesin-1/Kif5B, is essential for glucose-stimulated insulin secretion from pancreatic β-cells. 5΄AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase, which is activated in β-cells at low glucose concentrations, but inhibited as glucose levels increase. AMPK activation blocks glucose-stimulated recruitment of secretory granules to the cell surface and insulin secretion, suggesting motor proteins may be targets for this kinase. Whilst both kinesin-1/Kif5B and kinesin light chain-1 (KLC1) contain consensus AMPK phosphorylation sites only a peptide corresponding to Ser520 in mouse KLC1 and purified recombinant GST-KLC1 were phosphorylated by purified AMPK in vitro. To test the hypothesis that phosphorylation at this site may modulate kinesin-1-mediated granule movement, we developed a novel approach to study the dynamics of the granules within a cell in three-dimensions using Nokigawa spinning disc confocal microscopy. This cell-wide approach revealed that the number of longer excursions (>10 µm) increased significantly in response to elevated glucose concentration (30 vs 3 mM) in control MIN6 cells. However, similar changes were seen in cells over-expressing wild-type KLC1, phosphomimetic (S517/520D) or non-phosphorylatable (S517/520A) mutants of KLC1. Moreover, no evidence for a change in the phosphorylation state of KLC1 at Ser520 after AMPK activation was obtained using an anti-phospho Ser520-specific antibody. Thus, changes in the phosphorylation state of KLC1 at Ser517/520 are unlikely to affect motor function. In conclusion, we describe a new three-dimensional cell wide approach for the analysis of secretory granule dynamics in living β-cells.