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Defensores de Belgrano was founded on 25 May 1906 by a group of young people who wanted to participate in the football leagues of Buenos Aires. In 1914, Defensores de Belgrano won the División Intermedia champion, therefore promoted to Primera División, the top division. The squad debuted in Primera in 1915, being relegated to the second division at the end of that season. The team returned to Primera for the 1918 season, remaining there until 1934 when the official association (AFA) (which Defensores was affiliated to) merged with the dissident professional league (Liga Argentina de Football); as a result of the merger, all teams in the amateur league were relegated to the second division.

During its first years, the team's color's were light blue and light pink but would later be changed to the red and black colors that have remained since then. The stadium was at the "Plaza Alberti" venue in the suburb of Belgrano, Buenos Aires.

In 1934 two players of the club, Ernesto Belis and Luis Izetta went on to play for the Argentina national team in the 1934 FIFA World Cup held in Italy that year.

For many decades Defensores played the league games between the B and the C League's, gaining promotion and also being relegated. Throughout that period a rivalry against Platense was developed.

In August 2011, the veteran 37-year-old player Ariel Ortega (who had been played for All Boys during the 2010–11 season) was signed by Defensores de Belgrano. The "Burrito" made his debut in the match against Deportivo Morón scoring a goal by penalty shot.