Vitamin D and intramyocellular lipid (IMCL) both affect muscle function, but the relationship between vitamin D status and IMCL has not been established. To assess the relationship between vitamin D [measured as 25-hydroxy-vitamin-D (25(OH)D)] and IMCL, 20 community-dwelling adults between the ages of 65 and 85 were recruited. Serum 25(OH)D, and gastrocnemius IMCL and extramyocellular lipid (EMCL) were measured with magnetic resonance spectroscopy and fat ratio segmentation. A lifestyle questionnaire assessed physical activity. Muscle strength (1-repetition maximum) and physical function tests (timed up and go, timed sit to stand, four square step test, and gait speed) were also performed. Mean 25(OH)D was 37.9 ± 13.1 ng/mL with a range of 19-68 ng/mL. Soleus and gastrocnemius IMCL to water ratio was 1.04 ± 0.43 and 0.53 ± 0.22, respectively, but only gastrocnemius IMCL was correlated with 25(OH)D (R (2) = 0.39; p = 0.02). This relationship was independent of body mass index (p > 0.14), physical activity level (p > 0.08), and sex (p > 0.13). 25(OH)D did not correlate with EMCL (R (2) = 0.007; p = 0.78). The four square step test was the only performance or strength test correlated with 25(OH)D (R (2) = 0.26; p = 0.023). Muscle strength and physical function measures were not correlated with IMCL or EMCL. These data suggest that vitamin D status may influence gastrocnemius IMCL content independent of body mass and physical activity. Future studies should consider exploring whether vitamin D has an independent role in affecting muscle lipid metabolism and function.