This study evaluated the effects of varying levels of L-arginine (
Arg
) on performance and intestinal health of broilers challenged with
Eimeria.
Cobb 500 male chicks (
n
= 720) were randomly distributed in a 5 × 2 factorial arrangement (6 replicates/12 birds). The main factors were Arg levels (1.04, 1.14, 1.24, 1.34, 1.44%) and challenge or non-challenge with
Eimeria.
At day 12, in the challenge group, each bird received orally 12,500
Eimeria maxima
, 12,500
Eimeria tenella
, and 62,500
Eimeria acervulina
sporulated oocysts. At 5 d postinfection (
dpi
), intestinal permeability was measured. At 6 and 14 dpi, performance, intestinal histomorphology, nutrient digestibility, tight junction protein (
TJP
) gene expression, and antioxidant markers were evaluated. Few interactions were found, and when significant, the supplementation of Arg did not counteract the negative effects of
Eimeria
challenge. Challenge, regardless of Arg level, increased intestinal permeability, although the expression of Claudin-1, a TJP, was upregulated. At 6 dpi, the antioxidant system was impaired by the challenge. Moreover, growth performance, intestinal histomorphology, and nutrient digestibility were negatively affected by challenge at 6 and 14 dpi. Regardless of challenge, from 0 to 14 dpi, birds fed 1.44% showed higher weight gain than 1.04% of Arg, and birds fed 1.34% showed lower feed conversion than 1.04% of Arg. At 5 dpi, intestinal permeability was improved in birds fed 1.34% than 1.04% of Arg. Moreover, 1.34% of Arg upregulated the expression of the TJP Zonula occludens-1 (
ZO-1
) as compared with 1.24 and 1.44% of Arg at 6 dpi. At 14 dpi, 1.44% of Arg upregulated the expression of ZO-1 and ZO-2 compared with 1.24 and 1.34% of Arg. The nutrient digestibility was quadratically influenced by Arg, whereas the antioxidant markers were unaffected. Thus, the challenge with
Eimeria
had a negative impact on growth and intestinal health. The dietary supplementation of levels ranging from 1.24 to 1.44% of Arg showed promising results, improving overall growth, intestinal integrity, and morphology in broilers subjected or not to
Eimeria
challenge.