We evaluated the protective effects of probiotic administration as a prophylaxis treatment and immediately after fever onset in increasing the immune response and decreasing the renal scarring in a rat model of acute pyelonephritis. Twenty-four rats were apportioned to three groups. In GI (n = 8), the rats were injected with direct inoculation of Escherichia coli into the right kidney. In GII (n = 8), the rats received a probiotic regimen 1month before E. coli injection and the probiotic regimen was continued for the next 2months. In GIII (n = 8), the probiotic regimen was started just after E. coli injection and was continued for 2months. Technetium-99m-DMSA renal scan, histopathological evaluations, concentrations of CA19-9, IgA, blood urea nitrogen (BUN), and creatinine were assessed 1 and 2months post-injection. It took an average of 4.2 ± 1.1h between the injection and onset of fever in GI and GII. In GIII, this period was longer (7.5 ± 1.4). Probiotic administration resulted in reduction of interstitial fibrosis and tubular and glomerular atrophy in GII in all follow-ups. Technetium-99m-DMSA renal scan showed that the right kidney reached near the normal cortical integrity (47%) in GII compared to GI (32%) after 2months of injection. However, the renal integrity did not improve significantly in GIII (41%). In GII, CA19-9 was lower (p < 0.05), while the levels of serum and fecal IgA were higher (p < 0.05). Administration of the probiotic regimen in the rat model may decrease renal damage in pyelonephritis. In spite of better results in the prophylactic group compared to the treatment group, no strong evidence was found to prove the advantage of its prophylactic application over the treatment administration.