Successful introduction of therapeutic genes into the central nervous system (CNS) requires the further development of efficient transfer vehicles that avoid viral vector-dependent adverse reactions while maintaining high transfection efficiency. The multifunctional protein 249AL was recently constructed for in vitro gene delivery. Here, we explore the capability of this vector for in vivo gene delivery to the postnatal rat CNS. Significant transgene expression was observed both in the excitotoxically injured and noninjured brain after intracortical injection of the DNA-contaning-249AL vector. In the injured brain, a widespread expression occurred in the entire lesioned area and retrograde transport of the vector toward distant thalamic nuclei and transgene expression were observed. Neurons, astrocytes, microglia, and endothelial cells expressed the transgene. No recruitment of leukocytes, demyelination, interleukin-1β expression, or increase in astrocyte/microglial activation was observed at 6 days postinjection. In conclusion, the 249AL vector shows promising properties for gene therapy intervention in the CNS, including the targeting of different cell populations.