To develop novel cephem antibiotics, We have synthesized a new compound, named YH-487, by attaching the thiol and aminothiazole residue to C₃ and C_7 position of 7-ACA, respectively. Several characteristics such as structure, antibiotic spectrum, action mechanism, stability against β-lactamase and synergistic effect were investigated. Anti-bactericidal activity of YH-487 against gram-positive and gram-negative bacteria were superior to that of the other cephem antibiotics. We have examined the action mechanisms of YH-487 using penicillin binding protein (PBP) assay, and found that the bactericidal activity was obtained by inhibiting PBP-1A, PBP-1B and PBP-3. YH-487 showed synergistic effect with gentamicin, tobramycin, and amikacin against Pseudomonas aeruginosa. In addition, YH-487 was effective against Enterobacter cloacae in combination with amikacin. Based on the above observations, YH-487 was classified as a novel third-generation β-lactam antibiotics.