Abstract Background This study aimed to investigate the implications of antinuclear antibodies (ANAs) in juvenile idiopathic arthritis (JIA), the association with uveitis and the favorable evolution of the disease. Methods We followed 45 pediatric patients suffering from JIA, according to the International League of Associations for Rheumatology (ILAR), for a period of 2 years. ANAs were followed through immunofluorescence methods using a high-performance immunofluorescence automatic Helios device. Results A total of 15 of the ANA-positive patients presented an oligoarticular form of the disease, eight of them presented a polyarticular form of the disease and two of them presented a systemic form. The most severe forms have associated iridocyclitis. ANA-positive patients presented an earlier onset of disease in comparison with ANA-negative ones. ANA-positive patients from different ILAR categories had similar characteristics of the disease such as onset age (earlier), higher prevalence in females and the presence of asymmetric arthritis or iridocyclitis. Conclusions ANA may be an important marker for JIA and association with iridocyclitis. Following ANA titer in evolution might be a criterion for assessing the evolution of the disease. The association between positive ANA, oligoarticular JIA, iridocyclitis and a 2- to 4-year-old onset is common.