Neurofilament protein-L (NF-L) is the core component of neurofilaments. Recent studies indicate that the NF-L mutations reported in human Charcot-Marie-Tooth (CMT) disease lead to the formation of NF-L aggregates and result in axon degeneration of motor and sensory neurons, which are thought to be the cause of CMT disease type 2E. In the present study, we investigated the dynamic regulation of NF-L assembly and the mechanism of aggregate formation of CMT NF-L mutants. We report that 14-3-3 proteins interact with NF-L in a phosphorylation-dependent manner. Investigation of mutations of phospho-serine sites at the head domain of NF-L revealed that several phosphorylation sites, particularly Ser43 and Ser55, were important for 14-3-3 binding. 14-3-3 overexpression resulted in a significant increase in the dynamic exchange rate of NF-L subunits and induced striking disassembly of neurofilaments. CMT NF-L mutants, particularly those with mutations in the Pro8 and Pro22 sites of the NF-L head domain, led to substantially diminished interaction between 14-3-3 and NF-L, which resulted in the formation of NF-L aggregates and the disruption of the neurofilament co-assembly of NF-L and NF-M. However, aggregate formation in CMT NF-L mutants was downregulated by 14-3-3 overexpression. Taken together, these results suggest the important role of 14-3-3 in the dynamic regulation of NF-L assembly, and in the capacity to prevent the formation of NF-L aggregates. Thus, the 14-3-3 proteins are a possible molecular target for CMT disease therapy.