The endospore is the dormant form of Bacillus subtilis and many other Firmicutes. By sporulation these spore formers can survive very harsh physical and chemical conditions. Yet they need to go through germination to return to their growing form. The spore inner membrane (IM) has been shown to play an essential role in triggering the initiation of germination. In this study, we isolated the IM of bacterial spores, in parallel with the isolation of the membrane of vegetative cells. With the use of GeLC-MS/MS, over 900 proteins were identified from the B. subtilis spore IM preparations. By bioinformatics based membrane protein predictions, ca. one-third could be predicted to be membrane localized. A large number of unique proteins as well as proteins common to the two membrane proteomes were seen. In addition to the previously known IM proteins, a number of IM proteins were newly identified, at least some of which are likely to provide new insights into IM physiology unveiling proteins putatively involved in spore germination machinery and hence putative germination inhibition targets.