Introduction: The ageing process can be influenced by different dietary patterns and dietary factors. Aim: To analyse the association of dietary factors, which include the Mediterranean diet adherence, the compliance with the “Sociedad Espanola de Nutricion Comunitaria” food portion recommendations, and the intake of different food groups, with the secreted form of the alpha-Klotho gene (S-Klotho as an anti-ageing marker) in middle-aged sedentary adults. Methods: A total of 74 (39 women) middle-aged sedentary adults participated in the study. Dietary factors were assessed with a food frequency questionnaire and the PREDIMED questionnaire. The S-Klotho plasma levels were measured using a soluble alpha-Klotho ELISA assay kit. Results: We observed: (i) a negative association between the adherence to the Mediterranean diet and the S-Klotho plasma levels (beta = – 53.219; R-2 = 0.078; P = 0.020), (ii) a higher intake of wine (> 7 glasses/week), was associated with lower S-Klotho plasma levels (P = 0.039), and (iii) a positive association between nuts portion intake and the S-Klotho plasma levels (beta = 26.087; R-2 = 0.070; P = 0.029). All the associations disappeared after controlling for lean mass index (LMI) (all P > 0.05). Conclusion: Our study suggests that, although dietary factors could be related to S-Klotho plasma levels in middle-aged sedentary adults, LMI is the main determinant in this regard.