This cross-sectional comparative study aimed to compare serum L-carnitine and 25(OH)D levels between men with ED non-responding for oral sildenafil citrate and healthy volunteers. Overall, 192 men, recruited from two University Hospitals, were allocated into two equal groups of matched age; healthy potent men and men with ED non-responders for oral sildenafil citrate. Oral sildenafil citrate non-responders self-reported inadequate erectile responses after four attempts using 100 mg with the manufacturer's guidelines relative to meals, associated medications, and sexual stimulation/arousal. Exclusion criteria were: diabetes, cardiovascular disorders, beta blockers treatment, morbid obesity, thyroid disorders, post-radical prostatectomy, and hepatic/renal failure. All participants were subjected to; history taking, clinical examination, validated IIEF-5 questionnaire, estimation of serum L-carnitine by calorimetric method and serum 25(OH)D by ELISA method. Compared with potent controls, ED men non-responders for oral sildenafil citrate showed significant decreases in the mean serum L-carnitine level (16.8 ± 3.6 uM/L versus 66.3 ± 11.9 uM/L, P = 0.001), the mean serum 25(OH)D level (21.2 ± 7.1 ng/ml versus 54.6 ± 7.9 ng/mL, P = 0.001) and IIEF-5 score (7.8 ± 2.6 versus 23.9 ± 1.3). Serum L-carnitine showed significant positive correlation with IIEF-5 scores (r = 0.873, P = 001), serum 25(OH)D (r = 0.796, P = 0.001) and significant negative correlation with the age (r = -0.515, P = 0.001). Serum 25(OH)D showed significant positive correlation with IIEF-5 scores (r = 0.855, P = 0.001) and significant negative correlation with the age (r = -0.223, P = 0.005). It is concluded that normal homeostasis of serum L-carnitine and 25(OH)D play a role in male sexual health being significantly decreased in ED non-responding for oral sildenafil citrate.