Addiction to nicotine and the inability to quit smoking are influenced by genetic factors, emphasizing the importance of understanding how genes and drugs of abuse mechanistically impact each other. One well-characterized protein responsible for regulating both response to drugs and gene expression is the transcription factor CREB (cAMP-responsive element binding protein). Previous work indicates that hippocampal-specific alterations in CREB signaling and synaptic plasticity may underlie certain nicotine withdrawal phenotypes. However, the structure of the hippocampus possesses dorsal and ventral subregions, each differing in behavioral, anatomic and gene expression characteristics. This study examines the effects of CREB deletion specifically in the ventral or dorsal hippocampus of animals chronically treated with saline, nicotine, or undergoing 24 h withdrawal. After region-specific viral injections of AAV-GFP or AAV-CRE in CREBloxP/loxP animals, behavioral testing measured anxiety levels, using the Novelty-Induced Hypophagia test, and cognition, using a contextual fear conditioning paradigm. Deletion of CREB in the ventral, but not dorsal, hippocampus resulted in amelioration of nicotine withdrawal-induced anxiety-like behavior in the Novelty-Induced Hypophagia test. In contrast, CREB deletion in the dorsal hippocampus resulted in learning and memory deficits in fear conditioning, whereas CREB deletion in the ventral hippocampus showed an enhancement in learning. Gene expression analysis showed differential treatment- and region-dependent alterations of several CREB target genes that are well-known markers of neuroplasticity within the hippocampus. Collectively, these data provide persuasive evidence towards the distinct roles of CREB within the dorsal and ventral hippocampus separately in mediating select nicotine withdrawal phenotypes.