The aim of this study was to evaluate potential drug–drug interaction between lithium and levofloxacin. The study was conducted on New Zealand white rabbits with three groups having two subgroups each (n = 12). The first group compared the pharmacokinetic (Pk) parameters of lithium when lithium was given as a single dose (56 mg/kg) and when lithium was co-administered with levofloxacin (35 mg/kg). The second group compared the Pk parameters of lithium when lithium was given for 6 days alone and when levofloxacin was given on the sixth day after lithium steady-state levels were achieved. The third group compared the Pk parameters of lithium when lithium was given alone for 8 days and levofloxacin was given on days 6, 7, and 8 along with lithium. Apart from this, creatinine levels were also measured to detect nephrotoxicity effects because of this co-administration. It was found that there was increase in lithium levels in all three groups. The increase was significant when a single dose of levofloxacin was administered with steady-state level of lithium (Cmax of lithium: 2.54 ± 0.15 vs 2.79 ± 0.12 mm, P < 0.001 and AUC0-α of lithium: 24.36 ± 3.68 vs 31.88 ± 4.83 mmol/mL h, P < 0.001). The increase in lithium levels was also significant when levofloxacin was coprescribed for 3 days after lithium steady-state levels were achieved (Cmax increased from 2.72 ± 0.29 to 3.96 ± 0.29 mm, P < 0.001 and AUC0-α increased from 27.1 ± 4.96 to 42.64 ± 4.94 mmol/mL h). Levofloxacin increases lithium levels when they are co-administered, and this interaction might be clinically significant as they may be coprescribed.