In this study, we performed a phenotypic, molecular, and biochemical characterization of a new inhibitor-resistant SHV (IRS) β-lactamase, SHV-84, produced by a clinical Escherichia coli strain (INSRA4590) isolated from a patient admitted to Centro Hospitalar de Coimbra, Portugal in 1999. Antimicrobial susceptibility testing, isoelectric focusing for isoelectric point determination, identification of β-lactamase-encoding genes, extraction and purification of the enzyme, and determination of kinetic constants (Km, kcat) and inhibitor 50% inhibitory concentrations (IC50s) were conducted. The clinical strain expressed SHV-1 (pI 7.6) and SHV-84 (pI 7.4), which differs from SHV-1 by the amino acid substitution Lys234Arg. The SHV-84-producing transformant E. coli DH5α exhibited a β-lactam resistance phenotype similar to the clinical strain. SHV-84 showed lower affinity for penicillins (Km: 64 to 101 μM vs. 11 to 31 μM for SHV-1) and decreased catalytic activity against these antibiotics (kcat: 216 to 1,042 s⁻¹ vs. 220 to 1,937 s⁻¹ for SHV-1), lower affinity for cephalothin (Km: 169 μM vs. 40 μM for SHV-1), no hydrolysis of extended-spectrum cephalosporins, reduced susceptibility to clavulanic acid (IC50: 2.21 μM vs. 0.17 μM for SHV-1), and increased susceptibility to tazobactam (IC50: 0.03 μM vs. 0.15 μM for SHV-1). In conclusion, the Lys234Arg substitution alone is responsible for decreased susceptibility to β-lactamase inhibitors, corroborating previous molecular dynamic simulation results suggesting Arg234 induces a change in the positioning of the Ser130 side chain. The new blaSHV nucleotide sequence was submitted to the EMBL Nucleotide Sequence Database as blaSHV-84 with accession number AM087453.