Background: Proton pump inhibitors (PPIs) are widely prescribed for acid-related gastrointestinal disorders, with prolonged use becoming increasingly common. Recent evidence has raised concerns about potential renal complications, particularly acute interstitial nephritis (AIN), a reversible cause of acute kidney injury. This study evaluated the association between long-term PPI use and AIN and determined its prevalence among chronic users. Methods: A retrospective observational study was conducted at Sree Balaji Medical College and Hospital, Chennai, using 10,000 archived medical records (2020–2025). From these, 638 patients with documented long-term PPI exposure were identified, and 120 met inclusion criteria (age ≥18 years, PPI use ≥2 weeks to 6 months, no pre-existing chronic kidney disease). Patients on nephrotoxic drugs or with autoimmune disorders were excluded. Clinical, biochemical, urinary, inflammatory, and imaging data were analyzed. Associations were assessed using chi-square tests and logistic regression. Results: Of the 120 patients, 29 (24.2%) developed AIN. A significant association was found between PPI use exceeding six months and AIN occurrence (p = 0.013). Affected patients frequently presented with hematuria (45%), proteinuria (23.3%), and eosinophiluria (p < 0.001). Imaging revealed cortical scarring or renal enlargement in 71.7% of AIN cases, while inflammatory markers were significantly elevated. Omeprazole (29.2%) and lansoprazole (28.3%) were the most commonly implicated agents, although nephrotoxicity risk did not differ significantly between PPI types (p = 0.31). Withdrawal of PPIs with supportive therapy, including corticosteroids, resulted in full recovery in 82.75%, while 17.24% progressed to chronic kidney disease. Conclusion: Long-term PPI use is significantly associated with AIN, particularly with therapy beyond six months. Early recognition, routine renal monitoring in chronic users, and prompt drug withdrawal are essential to prevent progression to chronic kidney disease. These findings underscore the importance of cautious prescribing, physician awareness, and stronger pharmacovigilance regarding PPI safety.