Vertebrates displaying seasonal shifts in reproductive behavior offer the opportunity to investigate bidirectional plasticity in sensory function. The plainfin midshipman (Porichthys notatus) teleost fish exhibits seasonal, steroid-dependent plasticity in frequency encoding by eighth nerve auditory afferents. To what extent changes in afferent encoding are due to plasticity of the target auditory hair cells, inclusive of molecular mechanisms of auditory encoding, were previously unanswered. Evoked potentials recorded in vivo from auditory hair cells of the sacculus, the primary division of the inner ear used for hearing in this and many teleosts, revealed frequency-dependent plasticity in auditory hair cell function in both males and females. Auditory thresholds were lower in reproductive compared to nonreproductive animals with the greatest differences occurring at higher frequencies corresponding to high-energy harmonics of vocalizations. This plasticity correlated with seasonal fluctuations in circulating steroid levels suggesting that, like saccular afferents, plasticity at the hair cell level may be steroid-dependent. Large-conductance calcium-activated potassium (BKCa) channels in non-mammalian vertebrates determine the electrical resonant properties of auditory hair cells with higher expression levels coinciding with increased best frequency. In teleosts, the pore- forming !-subunit of BKCa channels is encoded by duplicate slo1 genes resulting from whole genome duplication at the origin of teleosts. In midshipman, duplicate slo1 genes were retained through tissue-specific subfunctionalization. Expression of at least one slo1 gene is significantly upregulated in the auditory epithelium of reproductive animals compared to non-reproductives, supporting the hypothesis that increased slo1 and BKCa channel expression facilitates higher resonant frequencies and decreased thresholds in hair cells during the reproductive season. In vivo manipulation of BKCa currents using either the broad-spectrum potassium channel antagonist tetraethylammonium chloride (TEA) or the BKCa channel-specific antagonist iberiotoxin (IbTx) induced frequency-dependent changes in auditory hair cell response thresholds on the order of naturally occurring seasonal plasticity. Reproductive animals treated with either antagonist had thresholds like those recorded in nonreproductives, consistent with the role of increased BKCa channel expression facilitating higher electrical resonance in non-mammalian vertebrate hair cells. We propose that changes in BKCa channel abundance are a primary mechanism for frequency tuning plasticity in auditory hair cells among vertebrates.