Conventional markers of kidney function that are familiar to clinicians, including the serum creatinine and blood urea nitrogen levels, are unable to reveal genuine injury to the kidney, and their use may delay treatment. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, and the predictive role and pathogenic mechanism of MIF deregulation during kidney infections involving acute kidney injury (AKI) are not currently known. In this study, we showed that elevated urinary MIF levels accompanied the development of AKI during kidney infection in patients with acute pyelonephritis (APN). In addition to the MIF level, the urinary levels of interleukin (IL)-1β and kidney injury molecule (KIM)-1 were also upregulated and were positively correlated with the levels of urinary MIF. An elevated urinary MIF level, along with elevated IL-1β and KIM-1 levels, is speculated to be a potential biomarker for the presence of AKI in APN patients.