CHRK1 is a receptor-like kinase containing a chitinase-related sequence in the extracellular domain in Nicotiana tabacum. The previous study indicated that CHRK1 plays a role in a signaling pathway regulating plant development and the endogenous cytokinin levels. In this study, we identified NtPUB4 as a CHRK1-interacting protein using yeast two-hybrid screening. NtPUB4 contains the U-box and five arm repeats, and is homologous to Arabidopsis AtPUB4 with unknown function and to Brassica arm repeat containing 1 (ARC1) that interacts with SRK receptor-like kinases during self-incompatibility response. The arm repeats of NtPUB4 are important for the interaction with CHRK1. CHRK1-NtPUB4 interaction was confirmed by in vitro binding assay using the recombinant proteins. NtPUB4 exhibited spatial and temporal expression patterns that are very similar to those of CHRK1. Finally, GFP and RFP fusion experiments demonstrated that both CHRK1 and NtPUB4 are localized at the plasma membrane in vivo. These results strongly indicate that NtPUB4 is an interacting partner of CHRK1 receptor-like kinase, and is likely involved in modulating the plant developmental signaling pathway mediated by CHRK1.