Objective <p>To investigate the effects of unilateral ureteral obstruction on renal pelvic peristalsis and pacemaker cells in neonatal rats.</p> Methods <p>Thirty-six 2-day-old Sprague–Dawley neonatal rats were randomly assigned to the partial unilateral ureteral obstruction (PUUO), complete unilateral ureteral obstruction (CUUO), or sham-operated groups (<i>n</i> = 12 per group). One week after surgery, renal pelvic puncture manometry was performed at graded perfusion rates to assess pelvic pressure and peristaltic activity. Pacemaker cell populations in the renal pelvis were evaluated using quantitative immunofluorescence.</p> Results <p>In sham-operated rats, renal pelvic pressure increased gradually with perfusion rate, accompanied by adaptive changes in peristaltic frequency and amplitude. PUUO resulted in elevated pelvic pressure and altered peristaltic patterns, characterized by an early compensatory increase followed by functional decline at higher perfusion rates. In contrast, CUUO caused a marked and sustained rise in pelvic pressure with complete loss of coordinated peristalsis. Immunofluorescence analysis demonstrated a progressive reduction in pacemaker cell density from the sham-operated to PUUO and CUUO groups.</p> Conclusions <p>Unilateral ureteral obstruction disrupts renal pelvic peristalsis in a severity-dependent manner. Alterations in peristaltic activity are associated with a reduction in renal pelvic pacemaker cells, suggesting their involvement in obstruction-induced peristaltic dysfunction. These findings provide functional insights into the pathophysiology of congenital hydronephrosis.</p>