Establishment of the rumen microbiome can be affected by both early-life dietary measuresand rumen microbial inoculation. This study used a 2 × 3 factorial design to evaluate theeffects of inclusion of dietary fat type and the effects of rumen inoculum from differentsources on ruminal bacterial communities present in early stages of the lambs? life. Twodifferent diets were fed ad libitum to 36 pregnant ewes (and their lambs) from 1 month pre-lambing until weaning. Diets consisted of chaffed lucerne and cereal hay and 4% molasses,with either 4% distilled coconut oil (CO) provided as a source of rumen-active fat or 4%Megalac®provided as a source of rumen-protected fat (PF). One of three inoculums wasintroduced orally to all lambs, being either (1) rumen fluid from donor ewes fed the PF diet;(2) rumen fluid from donor ewes fed CO; or (3) a control treatment of MilliQ-water. Afterweaning at 3 months of age, each of the six lamb treatment groups were grazed in spatiallyseparated paddocks. Rumen bacterial populations of ewes and lambs were characterisedusing 454 amplicon pyrosequencing of the V3/V4 regions of the 16S rRNA gene. Speciesrichness and biodiversity of the bacterial communities were found to be affected by thediet in ewes and lambs and by inoculation treatment of the lambs. Principal coordinateanalysis and analysis of similarity (ANOSIM) showed between diet differences in bacterialcommunity groups existed in ewes and differential bacterial clusters occurred in lambsdue to both diet and neonatal inoculation. Diet and rumen inoculation acted together toclearly differentiate the bacterial communities through to weaning, however the micro-biome effects of these initial early life interventions diminished with time so that rumenbacterial communities showed greater similarity 2 months after weaning. These resultsdemonstrate that ruminal bacterial communities of newborn lambs can be altered by mod-ifying the diet of their mothers. Moreover, the rumen microbiome of lambs can be changedby diet while they are suckling or by inoculating their rumen, and resulting changes in therumen bacterial microbiome can persist beyond weaning