The calcium/calmodulin-dependent protein kinase CCaMK forms a complex with its phosphorylation target CIP73 (CCaMK-interacting protein of 73 kDa). In this work, a homolog of the animal HSC/HSP70 interacting protein (HIP) was identified as an interacting partner of CIP73 in Lotus japonicus. L. japonicus HIP contains all functional domains characteristic of animal HIP proteins. The C-terminal STI1-like domain of L. japonicus HIP was found to be necessary and sufficient for interaction with CIP73. The interaction between CIP73 and HIP occurred in both the nuclei and cytoplasm in Nicotiana benthamiana leaf cells. The interactions between CIP73 and HIP and between CIP73 and CCaMK could take place simultaneously in the same nuclei. HIP transcripts were detected in all plant tissues tested. As nodule primordia developed into young nodules, the expression of HIP was down-regulated and the HIP transcript level became very low in mature nodules. More nodules were formed in transgenic hairy roots of L. japonicus expressing HIP RNA interference at 16 days postinoculation as compared with the control hairy roots expressing the empty vector. It appears that HIP may play a role as a negative regulator for nodulation.