Holland Jupiler League consists of 20 clubs, who play each other in a double round-robin league, with each club playing the other club home and away. Each club plays every other club once in the first half of the season before the league takes a winter break around the Christmas and New Year's holiday season. The second half of the season sees the same fixtures as the first half, with the stadiums changed, although the two halves are not played in the same order.

At the end of each season, the champion of the Eerste Divisie is automatically promoted to the Eredivisie. Eight other clubs enter the Nacompetitie, a promotion/relegation playoff that includes the 16th- and 17th-place clubs in the Eredivisie. The following teams qualify for the Nacompetitie:

The club with the best record in the "first period" of the season (after 8 rounds).
The club with the best record in the "second period" of the season (rounds 9–16).
The club with the best record in the "third period" of the season (rounds 17–24).
The club with the best record in the "fourth period" of the season (rounds 25–32).
The two remaining spots are filled at the season's end by the highest-placed clubs that have not already earned automatic promotion or qualified for the Nacompetitie.

If the club that wins a period has qualified for the Nacompetitie by winning a previous period, its place is filled by the next-best club in that period that has not already qualified. Usually, the clubs that qualify for the Nacompetitie turn out to be the 2nd- through 9th-placed clubs in the final table. Clubs in the Nacompetitie face each other in a knock-out system with the numbers 16 and 17 of the Eredivisie for two places in next season's Eredivisie.

Between seasons 1971/1972 and 2007/2009 teams could not relegate from the Eerste Divisie. From the 2009/2010 season onwards, one team has been relegated from the Eerste Divisie to the Hoofdklasse (then the main amateur league of Dutch football). From the 2010/2011 to 2015/2016 seasons, the KNVB introduced a third and highest amateur tier called Topklasse, and Hoofdklasse clubs have been able to get promoted to that new division.

The remainder of the 2019–20 season was cancelled amid the coronavirus disease 2019 (COVID-19) pandemic, thus there were no promotions or relegations. Before the cancellation, the KNVB met in December 2019 and ruled that promotion to the second division and relegation to the third were suspended until 2022–23, and that the lowest-ranked Eerste Divisie Jong team will exchange places with its highest-ranked Tweede Divisie counterpart. In June 2022, the KNVB decided that relegation from the Eerste Divisie will stay absent for an extended period of 5 to 10 years.

Before the 2008/2009 season, Hoofdklasse clubs could promote by obtaining a professional licence. However, only a club going bankrupt or losing their license could result in clubs leaving professional football. The last clubs leaving professional football in that way were FC Wageningen and VCV Zeeland in 1992, and more recently HFC Haarlem and RBC Roosendaal, who went bankrupt in January 2010 and June 2011 respectively. The most recent additions to the league were AGOVV Apeldoorn in 2003 and FC Omniworld in 2005, expanding the league to 19 and later 20 clubs. However, for the 2010/11 season, the league returned to 18 clubs, as HFC Haarlem went bankrupt and FC Oss were relegated to the newly formed Topklasse. The 2012/13 season ended with 16 teams after AGOVV and BV Veendam went bankrupt. Four teams have been added to bring the division back up to 20 teams in 2013. Achilles '29 has been promoted from the Topklasse with the reserve teams of Ajax, FC Twente and PSV being added as well.

Since the 2016/2017 season there is automatic relegation to the third-tier, semi-professional Tweede Divisie. Clubs in the Tweede Divisie had to announce in mid-season if they want to be eligible for promotion. Only if one of those clubs won the Tweede Divisie championship is a team relegated from the Eerste Divisie.