Germ-free (GF) mice have increased bone mass that is normalized by colonization with gut microbiota (GM) from conventionally raised (CONV-R) mice. To determine if innate immune signaling pathways mediated the effect of the GM, we studied the skeleton of GF and CONV-R mice with targeted inactivation of MYD88, NOD1 or NOD2. In contrast to WT and Myd88−/− mice, cortical bone thickness in mice lacking Nod1 or Nod2 was not increased under GF conditions. The expression of Tnfα and the osteoclastogenic factor Rankl in bone was reduced in GF compared to CONV-R WT mice but not in Nod1−/− or Nod2−/− mice indicating that the effect of the GM to increase Tnfα and Rankl in bone and to reduce bone mass is dependent on both NOD1 and NOD2 signaling.