hhLIM, a member of LIM protein family, has two LIM domains and plays an important role in gene regulation, cytoskeleton organization and cell hypertrophy. To understand the functional importance of hhLIM in cytoskeleton organization and muscle hypertrophy, hhLIM and its mutants of two LIM domains were constructed and function of each LIM domain and its interaction with actin were studied. GST pull down assay and immunofluorescence assays showed that LIM domain 2 at the C-terminus of hhLIM is critical for its interaction with actin. The mutant of the LIM domain 2 in which two important Cys are replaced by Ser lost the capacity of hhLIM to interact with actin. Mutation of the LIM domain 1 at the N-terminus of hhLIM impaired the capacity of hhLIM to interact with actin. F-actin cross-linking assay identified that hhLIM could make F-actin to cross-link into bundles by interaction between LIM domain 2 and actin. In conclusion, LIM domain 2 at the C-terminus of hhLIM plays a central role in F-actin polymerization and cytoskeleton stabilization, whereas the first LIM domain is essential for the nuclear localization of hhLIM.