Microbial transformation studies can be used as models to simulate mammalian drug metabolism. In the present investigation, biotransformation of celecoxib was studied in microbial cultures. Bacterial, fungal, and yeast cultures were employed in the present study to elucidate the metabolism of celecoxib. The results indicate that a number of microorganisms metabolized celecoxib to various levels to yield eight metabolites, which were identified by high-performance liquid chromatography diode array detection and liquid chromatography tandem mass spectrometry analyses. HPLC analysis of biotransformed products indicated that majority of the metabolites are more polar than the substrate celecoxib. The major metabolite was found to be hydroxymethyl metabolite of celecoxib, while the remaining metabolites were produced by carboxylation, methylation, acetylation, or combination of these reactions. The methyl hydroxylation and further conversion to carboxylic acid was known to occur in metabolism by mammals. The results further support the use of microorganisms for simulating mammalian metabolism of drugs.