The aim of the study was to characterize isolates of Salmonella enterica serovar Enteritidis (S. Enteritidis) obtained from humans and layer farms in Belgium collected during 2000-2010. Three periods were compared, namely (i) before implementation of vaccination (2000-2004), (ii) during voluntary vaccination (2005-2006) and (iii) during implementation of the national control program (NCP) for Salmonella including mandatory vaccination against S. Enteritidis (2007-2010). The characteristics compared across time periods were distributions of phage type and multiple-locus variable number tandem-repeat assay (MLVA). While PT4 and PT21 were predominantly isolated in Belgium in layers and humans before 2007, a significant reduction of those PTs was observed in both populations in the period 2007-2010. The relative proportion of PT4b, PT21c and PT6c was found to have increased considerably in the layer population since 2007. In the human population, PT8, PT1 and the group of 'other' PTs were more frequently isolated compared to the previous periods. When comparing the proportion of the predominant MLVA types Q2 and U2, no significant difference was found between the layer and human population in the three periods and between periods within each category (layer and human). A significant difference in isolate distribution among MLVA clusters I and II was found between human and layer isolates recovered during Period 3 and in the human population between Period 1 and 3. Results suggest that the association between S. Enteritidis in layers and the occurrence of the pathogen in humans changed since implementation of the NCP in 2007.