PurposeTo examine relationships between mesopic visual sensitivity measurements on microperimetry and macular inner and outer retinal layer (IRL and ORL) thicknesses in healthy younger, middle‐aged and older subjects.MethodsIn total, 154 healthy adults were divided into three age groups each with similar mean sensitivity. Regional retinal sensitivity (determined by mesopic fundus‐controlled microperimetry) and IRL (ganglion cell‐related layer) and ORL thicknesses were measured in the five subfields: central fovea (1 mm diameter) and the quadrants temporal, nasal, superior and inferior of a parafoveal ring of outer diameter 3 mm and inner diameter 1 mm. Relationships between regional sensitivity and corresponding IRL and ORL thicknesses were assessed through a univariate and multivariate linear regression model.ResultsVisual sensitivity means for each subfield differed across age groups (all p < 0.001). In each parafoveal ring quadrant, mean IRL thickness was reduced in the older eyes compared to the other groups (all p < 0.0001). In the inferior region, worse sensitivity was correlated with greater IRL thickness (p = 0.0207) in the middle‐aged group and with a thicker ORL (p < 0.0001) and thinner IRL (p = 0.0003) in the older eyes (R2 = 0.51). The slopes of regression lines relating sensitivity to IRL thickness and age group (p = 0.0027) or to ORL thickness and age group (p = 0.0020) differed significantly.ConclusionsThe relationship observed between mesopic visual sensitivity and retinal layer thickness varied with age. A worse sensitivity was related to a thicker macular IRL layer in middle‐aged eyes and to a thicker ORL and thinner IRL in older eyes.