Antimicrobial susceptibility testing of clinical isolates is a crucial step toward appropriate treatment of infectious diseases. The clinical isolate Francisella philomiragia 14IUHPL001, recently isolated from a 63-year-old woman with atypical pneumonia, featured decreased susceptibility to β-lactam antibiotics when cultivated in 5% CO2. Quantitative β-lactamase assays demonstrated a significant (P < 0.0001) increase in enzymatic activity between bacteria cultivated in 5% CO2 over those incubated in ambient air. The presence of β-lactamase genes blaTEM and blaSHV was detected in the clinical isolate F. philomiragia 14IUHPL001 by PCR, and the genes were positively identified by nucleotide sequencing. Expression of blaTEM and blaSHV was detected by reverse transcription-PCR during growth at 5% CO2 but not during growth in ambient air. A statistically significant alkaline shift was observed following cultivation of F. philomiragia 14IUHPL001 in both ambient air and 5% CO2, allowing desegregation of the previously reported effects of acidic pH from the currently reported effect of 5% CO2 on blaTEM and blaSHV β-lactamases. To ensure that the observed phenomenon was not unique to F. philomiragia, we evaluated a clinical isolate of blaTEM-carrying Haemophilus influenzae and found parallel induction of blaTEM gene expression and β-lactamase activity at 5% CO2 relative to ambient air. IMPORTANCE β-Lactamase induction and concurrent β-lactam resistance in respiratory tract pathogens as a consequence of growth in a physiologically relevant level of CO2 are of clinical significance, particularly given the ubiquity of TEM and SHV β-lactamase genes in diverse bacterial pathogens. This is the first report of β-lactamase induction by 5% CO2.