The aim of this study was to determine to what extent the ions present in hard water (125 mg/L of MgCl2 and 500 mg/L of CaCl2) may intensify the feed-induced decrease in oxytetracycline (OTC) absorption rate in broiler chickens after single oral administration at a dose of 15 mg/kg. Drug concentrations in plasma were determined by liquid chromatography-tandem mass spectrometry and combined, compartmental and non-compartmental approach was used to assess OTC pharmacokinetics. The administration of feed decreased the absolute bioavailability (F) of OTC from 12.70%±4.01 to 6.40%±1.08, and this effect was more pronounced after the combined administration of OTC with feed and hard water (5.31%±0.90). A decrease in the area under the concentration- time curve (AUC0-t), (from 10.18±3.24 μg·h/ml in control to 5.13 μg·h/ml±1.26 for feed and 4.26 μg·h/ml±1.10 for feed and hard water) and the maximum plasma concentration of OTC (Cmax) (from 1.22±0.18 μg/ml in control, to 1.01 μg/ml ±0.10 for hard water, 0.68 μg/ml±0.10 for feed and 0.61 μg/ml±0.10 for feed and hard water) was observed. The results of this study indicate that feed strongly decreases F, AUC0-t and Cmax of orally administered OTC. The ions present in hard water increase this inhibitory effect, which suggests that, therapy with OTC may require taking into account local water quality and dose modification, particularly when dealing with outbreaks caused by less sensitive microorganisms.