Mycobacterium tuberculosis (MTB) serine proteases are important pathogen-associated virulence factors that are involved in the invasion, bacterial persistence, and degradation of host defense factors. The current study identified and characterized a novel serine protease, Rv3194c, of MTB. A heterologous Rv3194c protein, purified from Escherichia coli, possessed proteolytic activity that could hydrolyze bovine serum albumin (BSA), milk, casein, and gelatin at an optimal temperature of 40 °C and a pH of 8.0. Furthermore, the divalent metal ions Ca²⁺ and Mn²⁺ increased the activity of Rv3194c. Betulinic acid, a Traditional Chinese Medicine (TCM) monomer; PMSF, a chemical inhibitor; and the Roche inhibitor cocktail inhibited proteolytic activity. Site-directed mutagenesis demonstrated that D308 and particularly S309 play a crucial role in the catalytic activity of Rv3194c protease. The cellular assays revealed that Rv3194c inhibits THP1-derived macrophage migration. Moreover, Rv3194c degraded the complement components, C3b and C5a, causing inhibition of phagocytosis and chemotaxis. In mice, Rv3194c enhanced the persistence of Mycobacterium smegmatis (Ms) in the lung, induced lung lesions, and promoted the release of inflammatory cytokines. The results of this study indicate that Rv3194c may play an important role in the pathogenicity of mycobacteria.