The presence of a conserved protein motif usually implies common functional features. Here, we focused on the LisH (LIS1 homology) domain, which is found in multiple proteins, and have focused on three involved in human genetic diseases; LIS1, Transducin beta-like 1X (TBL1) and Oral-facial-digital type 1 (OFD1). The recently solved structure of the LisH domain in the N-terminal region of LIS1 depicted it as a novel dimerization motif. Our findings indicated that the LisH domain of both LIS1 and TBL1 is essential for in vitro oligomerization. Furthermore, our study disclosed novel in vivo features of the LisH motif. Mutations in conserved LisH amino acids significantly reduced both the protein half-life of LIS1, TBL1, and OFD1, and dramatically affected specific intracellular localizations of these proteins. LIS1 mutated in the LisH domain induced its localization to the actin filaments. TBL1 mutated in the LisH domain was not imported into the nucleus. Mutations in OFD1 modified its localization to the Golgi apparatus and in some cases also to the nucleus. In summary, the LisH domain may participate in protein dimerization, affect protein half-life, and may influence specific cellular localizations. Our results allow the prediction that mutations within the LisH motif are likely to result in pathogenic consequences in genes associated with genetic diseases.