The existence of mild forms of celiac disease (CD) can make the histology-based diagnosis difficult to reach. Since anti-endomysium (EMA) and anti-tissue transglutaminase (anti-tTG) are detectable in culture supernatants of duodenal biopsies from CD patients, our aim was to assess if this system can support the histology in the diagnostic work-up. A total of 559 suspected CD patients underwent serum EMA/anti-tTG detection, upper endoscopy with duodenal biopsy sampling, histologic analysis, and organ culture to detect EMA/anti-tTG in supernatants. A subgroup of 30 patients with organ culture positive results were put on a gluten-free diet (GFD). Their gluten-dependency was evaluated by the psychological general well-being and beck depression inventory indexes. Statistical analysis was performed by Cohen k inter-test, Friedman test, and Dunn multiple comparison. Two hundred forty-one out of 559 (43.1%) patients showed intestinal villous atrophy, whereas serum and organ culture EMA/anti-tTG were positive in 293/559 (52.4%) and 334/559 (59.7%) patients, respectively. The strength of agreement resulted good for serology vs histology (k = 0.730), good for organ culture vs histology (k = 0.662), and very good for serology vs organ culture (k = 0.852). After 12 months of GFD, psychological general well-being index significantly increased, and beck depression inventory index significantly decreased (P < 0.001 for each one). Data highlight the organ culture system as a useful tool to assist the histology in diagnosing CD, mainly in cases without villous atrophy or in seronegative patients. The marked improvement in quality of life after a GFD further supports the reliability of this system in diagnosing CD.