Streptococcus pyogenes is an important human pathogen causing mild superficial infections of skin and mucous membranes, but also life-threatening systemic diseases. In this study we disrupted the arc-operon with the deletion of the arginine deiminase (AD) gene. The deletion had an impact on the amino acid and glucose metabolism but also on the virulence. Furthermore, the AD from S. pyogenes M49 was heterologously expressed in E. coli DH5α, purified, and kinetically characterized. The enzyme is well adapted to the environmental conditions in the human host. Therefore, the impact of the purified AD on cell proliferation of arginine auxotrophic tumors was investigated. The AD showed an antiproliferative effects on glioblastoma cell lines and hepatocellular and colorectal carcinoma cell lines. The antiproliferative effects were significantly increased in combination with chemotherapeutics.