<p>Our prior research demonstrated that the neuroprotective effects of clonidine can be reversed by yohimbine, an α2-adrenergic receptor antagonist. Additionally, clonidine has been shown to alleviate anxiety-like behavior in rats after bilateral common carotid artery occlusion by reducing the expression of proteins associated with hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels. However, the specific mechanisms by which clonidine inhibits HCN channels remain unclear. This study was designed to explore the protective effects of clonidine on neurons subjected to oxygen-glucose deprivation (OGD) injury and elucidate the underlying molecular mechanisms via HCN channels. The protective effects of clonidine on OGD-exposed neurons were confirmed by assessing the neuronal viability using a cell counting kit-8 (CCK-8) assay and by measuring the lactate dehydrogenase (LDH) release. Moreover, we identified the signaling pathways most relevant to clonidine’s action. PCR was performed to assess the PKA, AKT, HCN1, and HCN2 gene expressions, and a western blot assay was used to evaluate the related protein expressions of the AC–cAMP–PKA cascade, the PI₃K/Akt pathway, and the HCN channels. Clonidine and ZD7288 individually enhanced neuronal viability under OGD, demonstrating neuroprotective effects, with their combination yielding greater benefit. Clonidine upregulated α2A-AR and Nischarin protein levels. Its protection was attenuated by yohimbine (an α2-AR antagonist) and, to a lesser extent, by efaroxan (an I1R antagonist). KT5720, an AC–cAMP–PKA pathway inhibitor, synergized with clonidine and suppressed OGD-induced increases in HCN1 and HCN2 expression. Conversely, LY294002, a PI3K/Akt inhibitor, counteracted clonidine’s protection and further enhanced HCN1/HCN2 expression. These findings indicate that clonidine protects against OGD-induced injury mainly via α2-AR and partially through I1R, potentially by modulating HCN channels via the AC–cAMP–PKA and PI3K/Akt pathways.</p>