Exposure to cigarette smoke and ethanol are proposed to trigger neurotoxicity, apoptosis, and to impair neuronal signaling. However, it is little known how the combination of both might trigger astrogliosis and the morphological changes capable of affecting a differential susceptibility of hippocampal regions to these licit drugs. The present study investigated the chronic effects of exposure to cigarette smoke and/or ethanol on behavioral parameters, apoptosis, and alteration in immunoreactivity of glial fibrillary acid protein (GFAP) and S100β in the CA1, CA3, and dentate gyrus (DG) of the rat hippocampus. Adult male Wistar rats (n = 32) were divided into four groups: vehicle (VE, glucose 3% in water, 10 mL/kg), cigarette smoke (TOB, total 12 cigarettes per day), ethanol (ethanol, 2 g/kg), and cigarette smoke plus ethanol (TOB plus ethanol, total 12 cigarettes per day plus ethanol 2 g/kg) for 54 days. The groups were submitted to tail-flick, open-field, and inhibitory avoidance tasks. The results showed that ethanol per se worsened the short-term memory. The association between TOB and ethanol increased the immunoreactivity of cleaved caspase-3 in the CA3 and DG regions. The TOB plus ethanol group showed a lower immunoreactivity to GFAP in all regions of the hippocampus. In addition, ethanol and TOB per se also reduced the immunoreactivity for GFAP in the DG. Ethanol increased S100β immunoreactivity only in the DG. In conclusion, this study showed that only ethanol worsened short-term memory, and the DG became more susceptible to changes in the markers investigated. This evidence suggests that DG is more sensitive to neurotoxicity induced by cigarette smoke and ethanol.