The influence of the low-trans (<1% of total fatty acids) puff pastry margarine and the rest period between laminating on dough physical characteristics and puff pastry quality was investigated. Experiment was planed according to factorial plan 32 with independent variables: quantity of puff pastry margarine (35–55% based on flour, variation interval 10) and rest period (15–45 min, variation interval 15). The experiment involved two margarine samples (MLT1 and MLT2) of similar fat phase composition, but whose physical properties were, statistically speaking, significantly different. The results show that the pastry containing 35% of margarine MLT1 and allowing the rest period of 30 and 45 min has a lift of above 4.0, it is of minimal firmness and has a high specific volume (3.5 cm3 g−1). Having used puff pastry margarine MLT2, the highest quality of dough and pastry was achieved in the sample containing 55% of margarine and with the maximum rest period of 45 min. The sample mentioned showed an excellent lift of 5.3 and had a specific volume of 4.0 cm3 g−1, but it as well exhibited the maximal firmness of pastry. The application of puff pastry margarine MLT1 can result in a satisfactory quality of pastry, but it can also cause a reduction in the fat content by 7.5% and in the content of trans fatty acids by two times, while the energy value is reduced by 12% when compared with the pastry containing puff pastry margarine MLT2.