Antibiotic producing bacteria often express enzymes for auto-resistance that covalently modify, and thereby inactivate, the drug by O-phosphorylation, O-adenylation or N-acetylation. The hyg21 gene found in the hygromycin A biosynthetic gene cluster from Streptomyces hygroscopicus NRRL 2388 shows resemblance to antibiotic modifying O-phosphotransferases. In vitro phosphorylation assays showed that Hyg21 mono-phosphorylates hygromycin and its analogs specifically on the fucofuranose moiety using ATP, and to a lesser extent GTP, as phosphoryl donors. The kinetic parameters for phosphorylation were determined by HPLC analysis. The enzyme phosphorylates hygromycin, methoxyhygromycin and des-methylene hygromycin with average k_cat values of 2 s^-1 and K_m values of ~ 30 µM. Phosphorylation of C5”- dihydrohygromycin and C5”- dihydromethoxyhygromycin, which are produced in low amounts in the fermentation, was considerably slower, with k_cat values of 0.1 s^-1 and K_m of 8 – 10 µM. The turnover numbers of the non-natural C5”dihydro isomers of the above substrates, obtained by chemical reduction, were comparable with those of hygromycin and methoxyhygromycin. The optimal pH and temperature for the phosphotransferase activity were 7.5 and 30 ºC respectively. The phosphorylated hygromycin did not inhibit the growth of hygromycin-sensitive ΔtolC E. coli and Streptomyces lividans in a disc-diffusion assay, suggesting antibiotic phosphorylation by Hyg21 as a self-resistance mechanism in S. hygroscopicus. Allelic replacement of hyg21 in the S. hygroscopicus genome resulted in a > 90% decrease in the production of hygromycin.