24 teams participated in the race. Each team had seven riders, one more than the 2022 edition. All 15 UCI Women's WorldTeams were automatically invited. They were joined by 9 UCI Women's Continental Teams selected by organisers PMG Sport/Starlight. The teams were announced on 25 May 2023.
In May 2023, the route was announced by organisers PMG Sport/Starlight. The race started in Tuscany with an individual time trial, before heading north-west through the Emilia-Romagna, Piedmont, Liguria regions. After seven stages, the race transferred to Sardinia for the last two stages. The announcement of the route was criticised, taking place around 1 month prior to the event. The route itself was also criticised, with a drop in the total number of stages and stage length compared to previous editions.
As with the previous editions, the route required a waiver from the Union Cycliste Internationale, as Women's WorldTour races have a maximum race length of six days.
The first stage of the 2023 Giro d'Italia Donne featured an individual time trial (ITT) with a length of 4.4 kilometres (2.7 mi) around Chianciano Terme in Tuscany. Heavy rain and thunderstorms affected the stage, with multiple riders crashing including Mavi Garcia and Chloé Dygert of Canyon–SRAM - with Dygert still managing to set the fastest time. 2022 winner van Vleuten then beat Dygert, before the organisers called for a pause due to the weather. Resuming around 20 minutes later, Letizia Paternoster of Team Jayco-AlUla then beat van Vleuten's time by five-hundredths of a second.
With over 100 riders remaining, the organisers paused the stage again due to standing water on the roads and full storm drains. The stage was eventually cancelled on the grounds of rider safety using the Extreme Weather Protocol. van Vlueten called the stage "a lottery" and that it was good that the stage had been cancelled.
All times set were disregarded, and no classification jerseys were awarded. The race therefore started on stage 2 on Saturday 1 July.
The third stage was neutralised at 1 kilometre (0.62 mi) from the finish line for safety reasons. Times for the general classification were taken from that point, and the stage had no time bonuses.
Prior to the race, organisers PMG Sport/Starlight stated that they could not afford the €730,000 cost of TV coverage, which is required for the UCI Women's World Tour. Media reports suggested that without TV coverage, the race could be cancelled. Subsequently, agreement was made between the Italian Cycling Federation and Italian national broadcaster RAI to broadcast the race. CyclingNews reported that each stage would have "roughly one hour" of live coverage on RAI, Eurosport and Global Cycling Network.
^Frattini, Kirsten (2021-10-16). "A closer look reveals the inequity at Tour de France Femmes". cyclingnews.com. Retrieved 2022-02-28. Regarding, the number of days of competition during a stage race, the UCI sets the elite women's stage races at six days, unless an exemption is made by its Management Committee.