Bohuslav Sobotka said he was “disturbed by the increase in hateful attacks in Britain aimed at the citizens of EU member states” in a telephone call to his British counterpart.
The conversation took place after the murder of Zdeněk Makar, a 31-year-old Czech man, in Poplar, east London, on 21 September.
“The Czech government finds it unacceptable to see Czechs attacked because of their origin and being treated as second-class citizens,” Mr Sobotka said.
“Therefore I asked the British prime minister ... to let me know what measures her government will adopt to stop these hateful attacks.”
In a statement, Downing Street said Ms May had offered Mr Sobotka her sincere condolences following Mr Makar’s death, but added police did not consider the crime to have been motivated by racism.
"This particular incident was not considered to be a hate crime," the spokesperson said, adding that Ms May told her Czech counterpart, "the UK government condemned hate crime in the strongest way possible and it had no place in British society."
UK police said in July that racism surged before and after the 23 June referendum on Britain's membership of the EU. Anti-immigration rhetoric employed by the winning Leave campaign is thought to be a cause of the spike in hate crime, although police also attributed the rise to increased vigilance by officers and greater awareness among the public.
More than 3,000 incidents were reported to police across the country between 16-30 June, which is a 42 per cent increase on the same period last year, according to the National Police Chiefs’ Council.
The killing of Mr Makar follows the murder of a 40-year-old Polish man in August, which is being investigated as a possible hate crime, although police have said the motive is still not clear.
Two Polish officers joined British police in the Essex town of Harlow after two other Poles were attacked outside a pub on 4 September.
Earlier this month Ms May expressed “deep regret” over attacks on Polish citizens living in the UK in a phone call to Polish prime minister Beata Szydło.
A 29-year-old man has been charged in connection with Mr Makar's death and two others, a man aged 19 and a 16-year-old boy, have been bailed until early October pending further investigations.
Czech authorities record 37,000 Czech citizens currently working in Britain.
Downing Street said Ms May and Mr Sobotka, "agreed that the UK and the Czech Republic enjoyed excellent bilateral relations and that the relationship would continue to go from strength to strength, particularly in defence and commercial areas."
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