Rumen microbiotas are known to influence the fat deposition (
FD
) in sheep, but controversy over causality remains unresolved. Here, we performed microbiome-wide association studies (
MWAS
), microbiome genome-wide association analysis (
mbGWAS
) and bidirectional mendelian randomization (
MR
) analyses on 1,150 sheep with genotype data from whole-genome resequencing, 16S rRNA sequencing and multilevel FD-traits data. We quantified the proportion of individual variation in FD-traits explained by host genetics, rumen microbiota, and their interaction effects. We identified 32 rumen microbiota biomarkers including
Bifidobacterium
that were associated with FD-traits (P
adj
<0.05). Further, utilizing five MR methods, we identified eight causal associations between marker genera and FD-traits (P
adj
<0.05), including
Butyrivibrio
,
Olsenella
,
p-2534-18B5 gut group
,
Prevotellaceae UCG-003
, and
Pseudobutyrivibrio
causing forward causal effects on FD, and changes in
Flexilinea
and
Suttonella
induced by FD. To our knowledge, this is the inaugural attempt to employ MR in sheep to investigate the causal relationships between gastrointestinal microbiota and complex phenotypes, underscoring the potential for developing interventions related to adipose deposition in sheep from the perspective of the rumen microbiome.