<p>In previous studies, we highlighted the remarkable diversity of potassium transport systems in <i>Enterococcus faecalis</i>. This microorganism encodes five distinct potassium transport systems: the Kdp complex (KdpFABC and KdpED), the Ktr systems (KtrAB and KtrAD), and transporters from the Kup family, specifically Kup and KimA. Research focused on the <i>E. faecalis</i> JH2-2 strain, which, unlike <i>E. faecalis</i> V583, lacks the Kdp complex and exhibits an altered expression of the <i>kup</i> gene due to the insertion of the IS6770 sequence. Here, we characterize the <i>kimA</i>-<i>kup</i> locus of the JH2-2 strain, which harbors genes encoding a metal transport protein (MntE), the Opu osmoresistance system (OpuCA-CB-CC-CD), and a Ca²⁺-ATPase (CaaT). It was determined that the <i>kimA</i> gene is monocistronic and transcribed from a consensus vegetative promoter featuring conserved -10 (TAgcAT) and -35 (TTGACt) boxes. RT-qPCR analysis revealed that <i>kimA</i> expression was significantly upregulated under acidic stress. Furthermore, both <i>kimA</i> and <i>kup</i> expression are increased under alkaline conditions in the presence of NaCl. To dissect functional roles, growth parameters of mutant strains KimA⁺ (Δ<i>ktrA</i>Δ<i>kup</i>) and Kup⁺ (Δ<i>ktrA</i>Δ<i>kimA</i>) were analyzed. Kinetic assays demonstrated that the Kup⁺ strain exhibited higher potassium transport affinity than KimA⁺. Furthermore, differential responses to pH and osmotic stress highlighted distinct physiological contributions of KimA and Kup systems. KimA seems to be involved in K⁺ uptake at high concentrations (&gt; 0.5&#xa0;mM) while Kup played a dominant role at low potassium concentrations (&gt; 0.2&#xa0;mM) and under alkaline-osmotic stress. All these results delineate the functional divergence of the two transporters and our understanding of the ion homeostasis and stress adaptation mechanisms in <i>E. faecalis</i>.</p>