Presence of excreted antibiotics in wastewater or manure may select for antibiotic resistant bacteria (ARB) by providing a selective environment. It is important, therefore, that antibiotics are inactivated in sewage treatment plants or manure handling facilities before being released into the environment. We evaluated the effect of three different concentrations (10, 50, and 100 mM) of three metal ions (Ferrous, Ferric and, Calcium) on the antimicrobial activity of tetracycline in water. Antimicrobial activity of metal ion-tetracycline solution was tested against Escherichia coli ATCC 25922, a tetracycline sensitive strain (MIC < 2 mg ml-1). In this procedure, 1 ml of antibiotic solution (500 mg ml-1) was mixed with 9 ml of metal ion solution. After overnight incubation, 1 ml of this solution was mixed with 4 ml of 3% tryptic soy broth (final tetracycline concentration 10 mg ml-1). This solution was inoculated with 100 ml of an overnight culture of E. coli followed by overnight incubation at 370C. Serial dilutions of the culture were plated on McConkey agar plates to enumerate bacteria. Appropriate negative and positive controls were also used. The numbers of E. coli in the presence of tetracycline and metal ions were compared with those in tetracycline alone. The antimicrobial activity of tetracycline was inactivated by metal ions and this effect was more pronounced in the presence of 50 mM Ferrous ions. Liquid Chromatography-Mass Spectroscopy (LC-MS) studies showed that tetracycline formed a complex with Ferrous ions, making it difficult to enter the cell through the cell wall and inhibit protein synthesis. These results indicate that Ferrous Chloride solution can be used for inactivation of tetracycline antibiotic in wastewater treatment plants or manure lagoons.