The Division Two side have suffered a difficult few months since joining the league with points deductions for fielding an ineligible player and failing to fulfil two fixtures in August.
Their record will be expunged in due course.
In a letter to the league, Barnoldswick Town chairman Nigel Hill talked of his 'great sadness' at withdrawing their reserve side from the league, adding "Our apologies go out to all your member clubs that we could not fulfil our season."
Barnoldswick Town's first team play in the North West Counties League.
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