The objective of this study was to determine the effect of dietary probiotic Pediococcus acidilactici (PA) strain MA18/5M on performance, egg traits, egg cholesterol content, and fatty acid composition in laying hens during a 24-wk period. A total of 222 Hy-Line Brown laying hens, 22 wk of age, were divided into 3 treatment groups. Control group (C) hens were fed a basal diet with no probiotic added. In group PA1, the basal diet was supplemented with PA at 100 mg.kg(-1) of feed for the first 12 wk and 50 mg.kg(-1) feed for the next 12 wk, whereas treatment PA2 was supplemented with 100 mg.kg(-1) feed for the whole trial period. Dietary treatments did not significantly affect the BW, feed intake, and egg production of hens. Pediococcus acidilactici supplementation increased egg weight (P < 0.05), eggshell thickness, eggshell relative weight, and egg specific gravity, and it improved feed efficiency ratio per kilogram of eggs (P < 0.01). Moreover, PA dietary supplementation resulted in a significant (P < 0.05) decrease in the number of broken eggs and eggs without the shell, leading to a significant (P < 0.01) reduction in the number of downgraded eggs (39% for PA1 and 52% for PA2). After 6 mo of probiotic supplementation, significant differences were also found in the fatty acid composition and cholesterol content of egg yolk. The yolk cholesterol content, regardless of PA dose, decreased by more than 10%. The concentrations of total polyunsaturated fatty acids, including linoleic acid and linolenic acid, were significantly higher in treatment PA2 (6.5% increase) than in C and PA1. In conclusion, dietary supplementation of Pediococcus acidilactici MA 18/5M at 100 mg.kg(-1) has potential commercial applications for improvements in hen performance and eggshell quality during the early laying period.