Background/Objectives: 11β-Hydroxysteroid dehydrogenase (11β-HSD) enzymes convert cortisol into inactive cortisone and vice versa. While 11β-HSD type 2 (mainly localized in the kidney) unidirectionally inactivates cortisol to cortisone, type I isoform (mainly localized in the liver) acts bidirectionally and can thus potentially restore cortisone to active cortisol. The aim of this pilot study was to investigate whether the serum cortisol:cortisone ratio is altered during the acute-phase response, possibly due to altered modulation of 11β-hydroxysteroid dehydrogenase isoforms. Methods: Using liquid chromatography electrospray tandem mass spectrometry, cortisol and cortisone were measured in the serum of hospitalized patients with normal and abnormal CRP concentrations, the latter indicating acute-phase response. Fifteen unselected samples were analyzed, all with a CRP concentration within one of the following ranges to cover a wide range of CRP concentrations evenly: <5, 5–20, 21–50, 51–100, 101–200, and >200 mg/l. Results: In the heterogeneous study population, increased CRP concentrations significantly correlated with an increased cortisol:cortisone ratio (p < 0.001; r = 0.65, Spearman correlation coefficient). This correlation was independent of increased serum cortisol concentrations found by multivariate regression analysis. The median ratio was 6.4 (interquartile range 5.5–7.4; n = 30) in patients with a CRP concentration ≤20 mg/l, and 11.2 (interquartile range 8.8–13.9; n = 60) in patients with CRP >20 mg/l (p < 0.01). Conclusion: The balance between serum cortisol and cortisone is altered during acute-phase response with a shift towards active cortisol, suggesting that 11β-HSD isoenzymes play a role in the modulation of systemically available cortisol during acute illness.