<b><i>Background:</i></b> Acute kidney injury (AKI) is a frequent condition, with persistent shortage of large-scale epidemiological studies. We analyzed the population-wide healthcare system of the Italian Lombardy region over the 2000–2019 period, and evaluated AKI incidence, mortality, and related healthcare resource utilization and cost in all citizens 40 years and older. <b><i>Methods:</i></b> The retrospective cohort analysis of an administrative claims database that routinely collects information about healthcare provision in a high-income region with 10 million citizens. Over 20 years, AKI was identified in 84,384 hospital discharge records by the International Classification of Diseases 9th Revision codes (mean age 77.4 ± 11.6 years, 52.5% were males). <b><i>Results:</i></b> From 2000 to 2019, the AKI rates per 100,000 population changed from 32.9 to 90.5 for incidence, from 4.7 to 11.9 for mortality, and from 32.3 to 44.1 for years of life lost (YLLs), respectively. In-hospital mortality changed slightly (14.2% and 13.2%, respectively), while 30-day mortality decreased from 21.5% to 17.4%, respectively. Incidence rates increased with age and were higher in males, and varied almost four-fold between provinces. The median hospitalization cost was €4,014 (IQR: 3,652; 4,134), and the annual cost of treatment risen from €5.2 million in 2000 to €22.9 million in 2019. Hemodialysis was administered in 7.4% of hospitalizations. Over the total study period the cumulative AKI burden accounted for 11,420 in-hospital deaths, 63,370.8 YLLs, and €329 million of direct cost. <b><i>Conclusions:</i></b> This real-world analysis demonstrates the high burden of AKI with prominent geographical differences that require further implementation of preventive and diagnostic actions.