Production of L-glutamate oxidase (GluOx) by Streptomyces sp. N1 was investigated by controlling culture pH at 6.2, 6.7, 7.0, and 7.3 in a 5-L stirred fermenter. The corresponding GluOx activities obtained were 2.8, 4.2, 6.0, and 5.3 U/mL, respectively. Microbial growth was inhibited by increasing the medium pH from 6.2 to 7.0. The inhibitory effect was also observed in plate colony growth under incubation with a different initial pH value. The effect of calcium on GluOx production was also studied in the pH-controlled bioreactor. When the culture pH was controlled at 6.2 or 7.0, GluOx production could not be improved or was only improved slightly by initial addition of calcium to the medium. However, when the culture pH was kept at 6.7, initial Ca2+ addition (60 mM) conspicuously enhanced GluOx production up to 9.3 U/mL, which was about twofold of that without Ca2+ addition. The enzyme production level was the highest ever reported in the literature. During fermentation the inhibition of cell growth by Ca2+ addition was observed. For the morphological changes, the cells mostly existed as pellets in the medium without Ca2+ addition, whereas few pellets were found and almost all the cells were dispersed mycelia in the broth with Ca2+ addition.