The ability to effectively oxidise intramuscular triglycerides (IMTG) contained within lipid droplets (LD) during exercise is considered to be a vital aspect of maintaining skeletal muscle insulin sensitivity in endurance trained athletes. The mechanism of IMTG lipolysis during exercise however, is not fully understood. Hormone sensitive lipase (HSL) is a key enzyme in IMTG lipolysis during exercise. Perilipin-5 (PLIN5) & Perilipin-2 (PLIN2) are abundant members of the LD proteome that have also been implicated in the regulation of IMTG lipolysis as PLIN2-LDs and PLIN5-LDs are preferentially oxidised during exercise. The aim of this study was to test the hypothesis that HSL translocates to PLIN5-LD and PLIN2-LD during moderate intensity exercise in human skeletal muscle. Percutaneous biopsies from the m. vastus lateralis of eight trained men (21±1 years, body mass index 22.6±1.2 kg.m-2 and peak oxygen uptake 48.2±5.0 ml.min-1.kg-1) were obtained pre and post 60 min of moderate-intensity exercise (∼65% VO2peak). Cryosections (5µm) were stained using antibodies targeting HSL, PLIN5 and PLIN2. LD were stained using BODIPY 493/503. Images were acquired using confocal immunofluorescence microscopy whereby fifteen fibres per time point per participant were obtained for colocalisation analysis using a 63x objective and 16x digital magnification. Values are means ± SEM. From pre- to post-exercise HSL re-organised from a small number of large clusters to a regular punctate distribution organised uniformly throughout the cytosol. During exercise HSL translocated towards the LD resulting in an increase in the amount of LD that were colocalised to HSL from pre- to post-exercise (55.6±1.7 % and 66.1±3.2 %, respectively; P<0.05, paired t-test). PLIN5-LD in comparison to PLIN5-null-LD had a higher increase in HSL colocalisation from pre-exercise (PLIN5-LD: 0.0041±0.0004 µm-2 and PLIN5-null-LD: 0.0015±0.0002 µm-2) to post-exercise (PLIN5-LD: 0.0063±0.0007 µm-2 and PLIN5-null-LD 0.0020±0.0002 µm-2; 2-way within-subjects ANOVA; P<0.01). Both PLIN2-LD and PLIN2-null-LD showed an increase in HSL colocalisation from pre-exercise (PLIN2-LD: 0.0057±0.0004 µm-2 and PLIN2-null-LD: 0.002±0.0002 µm-2) to post-exercise (PLIN2-LD: 0.0067±0.0003 µm-2 and PLIN2-null-LD: 0.0026±0.0005 µm-2; 2-way within-subjects ANOVA; P<0.05), however there was no main interaction effect (P=0.611). This study presents the first evidence of exercise-induced HSL translocation to LD in human skeletal muscle and identifies PLIN5 as a key facilitator of this mechanism. By identifying this additional step in IMTG lipolysis in healthy trained men, further research should aim to detect if dysregulation of this process occurs in sedentary, obese individuals which may contribute towards the development of insulin resistance.