Roughly one-third of patients with epilepsy remain drug resistant, underscoring the need for novel therapeutic strategies. Although cannabidiol (CBD) has recently been approved for specific epileptic encephalopathies, its anticonvulsant mechanisms and the influence of biological sex on treatment response remain incompletely understood. In this preclinical study, we evaluated the efficacy, tolerability, pharmacokinetics, and molecular effects of CBD in the GASH/Sal hamster, a genetic model of audiogenic generalized tonic–clonic seizures. Animals received intraperitoneal CBD (200 mg/kg) either acutely or chronically for 14 days. CBD concentrations were measured in serum and brain, while seizure severity, latency, and neuroethological parameters were assessed following acoustic seizure induction. Safety was evaluated through body weight, hematological, and biochemical analyses, and gene expression profiling was performed in the inferior colliculus, the primary epileptogenic focus. CBD achieved measurable systemic and brain exposure after both acute and chronic administration, despite substantial inter-individual variability and no significant sex differences in drug concentrations. CBD reduced audiogenic seizure severity in a time-dependent manner, with greater protection after chronic treatment. CBD also prolonged latency to seizure onset in both sexes (earlier in females). Although complete seizure suppression was more frequent in females than males (37.5% vs. 12.5% after chronic treatment), direct sex comparisons did not reach statistical significance. Notably, higher serum and brain CBD concentrations were associated with lower seizure severity. Chronic CBD administration was well tolerated in both sexes, affecting selected hematological parameters without altering body weight or liver function. Gene expression profiling revealed that transcriptional organization of the inferior colliculus was driven predominantly by biological sex rather than by seizure induction or CBD treatment. Marked sex-dependent differences were observed in serotonergic, endocannabinoid, purinergic, and Sigmar1-related neuroprotective pathways. Within this molecular context, seizure stimulation modulated Trpv1 and Slc29a1, whereas chronic CBD induced pathway-specific, sex-dependent changes involving 5-Htr1a, Adora1, and Cnr1, without eliciting widespread transcriptional remodeling. Collectively, these findings identify CBD as a well-tolerated anticonvulsant in the GASH/Sal model and suggest that pharmacokinetic exposure and the sex-specific molecular organization of the epileptogenic focus contribute to variability in treatment response, highlighting the importance of considering biological sex in cannabinoid-based epilepsy research.