Rome had earlier said it "does not accept ultimatums or impositions from abroad regarding national security and border control."
Spain is set to introduce retaliatory border controls on travellers from Italy amid a deepening dispute with Rome following last week's Ceuta migrant crisis.
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The new checks will begin at midnight on Saturday and last until September 7, Spain's Interior Ministry said in a statement, adding that it was a response to "the persistent irregular migration pressure" facing Italy.
The Spanish government had issued an ultimatum to Italy earlier on Friday, demanding that it lift border controls imposed on travellers from Spain by Sunday, arguing that the measure is "unjust, contrary to the interests of the European Union and discriminatory towards the Spanish population."
If the measures remain in place, Prime Minister Pedro Sánchez's administration warned that it would adopt "proportionate measures" to defend the interests of Spanish citizens, but it gave no details about what those measures might be.
In a statement, the Spanish government argued that the decision taken by Rome on 1 August was "based on spurious arguments incompatible with the Schengen Borders Code and with the facts."
Rome responded later on Friday, saying: "Under no circumstances do we intend to review our decision to suspend the application of the Schengen Agreement for third-country nationals coming from Spain, at least until 15 August" - when another mass migrant crossing into Ceuta is rumoured to be planned.
"Italy does not accept ultimatums or impositions from abroad regarding national security and border control," the Italian government continued in a statement, adding that the decision had been made "without prejudice" and that it was "in line with similar decisions" taken previously.
Italy justified the temporary reintroduction of checks after the mass arrival of migrants in Ceuta, although the inspections mainly affect air and sea traffic between the two countries.
The Spanish government points out that none of the migrants who entered Ceuta irregularly last week were allowed to move freely within the Schengen area, as the autonomous city has a special status.
It also stressed that the vast majority have already returned to Morocco and rejected the idea that this migrant crisis poses any risk to the other EU member states.
Spain also noted that, according to Frontex data, Italy has recorded in recent years the highest number of irregular crossings of the European Union's external borders, in some periods double Spain's figures.
It also criticised the Italian government for breaching the Dublin Regulation since 2022, shifting greater migratory pressure onto the rest of its European partners.
The government further underlined that Spain never responded with border controls despite that situation and recalled that it showed solidarity with Italy by taking in some of the migrants who arrived in Lampedusa in 2023 and by repeatedly opening its ports to people rescued on the Central Mediterranean route.
The government expressed confidence that "commitment to the European project, common sense and good faith" will prevail and accuses the Italian government of stoking "divisions and pointless conflicts driven by domestic electoral motives."