Oxidative damage and inflammation are two highly important factors that influence the development of cancer and other degenerative diseases. In this work, we explored the biological functions of a purified 25 kDa protease (SH21) from Bacillus siamensis. SH21 showed highly strong antioxidant and reactive oxygen species (ROS) generation inhibition actions in a dose-dependent manner. In the sample with SH21, the mRNA and protein levels of antioxidant enzymes such as superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione peroxidase 1 (GPx-1) were increased. NF-E2-related factor 2 (Nrf2)\'s transcriptional and translational activity was also boosted by SH21, which led to the development of the detoxifying enzyme heme oxygenase-1 (HO-1). Additionally, SH21 displayed an anti-inflammatory effect by reducing the production of proinflammatory cytokines like TNF-, IL-6, and IL-1 in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Nitric oxide synthase (iNOS) and cytokine gene expressions may have been reduced by SH21 at concentrations of 60, 80, and 100 g/mL. Furthermore, in a concentration-dependent way, SH21 dramatically produced the enzyme lactate dehydrogenase (LDH) in the cancer cell supernatant and showed potent activity against the three cancer cell lines HL-60, A549, and Hela. Our findings imply that SH21 has potent antioxidant, anti-inflammatory, and anticancer activities and may be a great treatment option for disorders caused by inflammation.