Impairment of learning and memory is a significant pathological feature of many neurodegenerative diseases including FTLD-TDP. Appropriate regulation and fine tuning of spinogenesis of the dendrites, which is an integral part of the learning/memory program of the mammalian brain, are essential for the normal function of the hippocampal neurons. TDP-43 is a nucleic acid-binding protein implicated in multi-cellular functions and in the pathogenesis of a range of neurodegenerative diseases including FTLD-TDP and ALS. We have combined the use of single-cell dye injection, shRNA knockdown, plasmid rescue, immunofluorescence staining, Western blot analysis and patch clamp electrophysiological measurement of primary mouse hippocampal neurons in culture to study the functional role of TDP-43 in mammalian spinogenesis. We found that depletion of TDP-43 leads to an increase in the number of protrusions/spines as well as the percentage of matured spines among the protrusions. Significantly, the knockdown of TDP-43 also increases the level of Rac1 and its activated form GTP-Rac1, a known positive regulator of spinogenesis. Clustering of the AMPA receptors on the dendritic surface and neuronal firing are also induced by depletion of TDP-43. Furthermore, use of an inhibitor of Rac1 activation negatively regulated spinogenesis of control hippocampal neurons as well as TDP-43-depleted hippocampal neurons. Mechanistically, RT-PCR assay and cycloheximide chase experiments have indicated that increases in Rac1 protein upon TDP-43 depletion is regulated at the translational level. These data together establish that TDP-43 is an upstream regulator of spinogenesis in part through its action on the Rac1 → GTP-Rac1 → AMPAR pathway. This study provides the first evidence connecting TDP-43 with the GTP-Rac1 → AMPAR regulatory pathway of spinogenesis. It establishes that mis-metabolism of TDP-43, as occurs in neurodegenerative diseases with TDP-43 proteinopathies, e.g., FTLD-TDP, would alter its homeostatic cellular concentration, thus leading to impairment of hippocampal plasticity.