The car parts and bicycles retailer posted a 4 per cent drop in like-for-like bike sales in the 13 weeks to 1 July.
A stronger performance from its car maintenance and travel solutions ranges failed to offset the bike disappointment, with overall first-quarter retail sales 1.2 per cent lower.
But Halfords said that, with the timing of Easter stripped out, retail sales were down a more modest 0.2 per cent.
It marks a setback in the revival of its bike sales, with Halfords having battled to turn around after a dire performance last summer left full-year cycle sales 0.9 per cent lower.
But the chain said it had faith in a decent peak summer cycling season ahead of the Rio 2016 Olympic Games, with new ranges from British hopeful Laura Trott and 2012 Tour de France winner Sir Bradley Wiggins.
Chief executive Jill McDonald said: “We had good sales growth of premium bikes, but cycling sales across the quarter were impacted by both the timing of Easter and poor weather, particularly in April. We look forward to the peak summer cycling season.”
She added that the Brexit vote had created “uncertainty” and the group cautioned that further falls in the value of the pound against the US dollar could hit profits further.
The group revealed in early June it expected a £3 million knock to profits from the weak pound, which has hit 31-year lows since the EU referendum decision, and said in its latest update that ongoing falls in the pound “may have a small further impact later in this financial year”.