His first destination as a police officer was Genoa.

In Rijeka, his second location, Palatucci was assigned to the foreigners' office, which was to be crucial for his future work as police commissioner.

Giovanni Palatucci, who was the head of the foreigners' office at the Rijeka Police Headquarters, following the enactment of the racial laws in 1938, did his utmost to bring help to foreign Jews who asked to enter Italy through the Rijeka Pass. In this way, he was able to prevent the deportation to Italian internment centres of Jews who were in Rijeka, either as residents or in transit. When this was not possible, he tried to have them sent to the Campagna (Salerno) internment camp, which was located in the diocese of his uncle who was bishop, knowing that the living conditions of the internees would be alleviated by his uncle's actions, thanks also to the hospitality of the local population. 
After 8 September 1943, the Germans took possession of Rijeka, and Palatucci became vice-police commissioner, continuing to help Jewish refugees, even saving them from deportation to the camps that existed in Italy at the time.

As he came under increasing suspicion by the German military authorities, by order of Gestapo Lieutenant Colonel Herbert Kappler, he was imprisoned on the night of 12-13 September 1944 on charges of collaborating with the enemy.

Locked up for about a month in the Coroneo prison in Trieste, he was then transported to the KZL (Konzentrationslager) in Dachau, where he arrived on 22 October 1944.
He died on 10 February 1945 due to the typhus epidemic that had been raging in the camp since the previous December and was buried in the mass grave on the Leitenberg hillock, about one kilometre from the concentration camp.
Recognitions of his work came a few years later, initially from the Jewish side, then also from the Italian side. In 1990, Yad Vashem in Jerusalem awarded him the title 'Righteous Among the Nations'.
However, caution must be exercised around the figure of Palatucci in discerning between apologia and truth. The many accounts of his work on behalf of Jews come from Jewish institutions, people he saved, his family and various institutions. According to some sources, he helped save some 5,000 Jewish refugees. Other research has downgraded this data, also noting that Palatucci would in no way have been in a position to help so many persecuted people.

The most comprehensive challenge to the reconstruction of Palatucci's story was made in May 2013 by a research study carried out by Primo Levi Center in New York, whose findings received some coverage in the international press, which questioned not only the scale of his involvement, but his actual contribution to the cause of Jewish refugees.