Paper is a hygroscopic material, and its application in classic offset printing creates additional challenges, especially in multi-color printing. The change in the size of the paper, due to the absorption of the wetting solution, reduces the quality of the print, and can affect the readability of the text and create a moiré effect. The operators behind the printing machines reduce deviations in the register with their interventions if the print would remain high-quality, but this requires knowledge and experience. The aim of this work was to determine the influence of the properties of the paper substrate on its behavior in multi-color printing. The research uses paper samples of different grammages, printed motifs with greater or lesser color application and different direction of fibers in the paper. The used samples were printed on a Heidelberg XL-106-8-P printing machine, which has the possibility of double-sided printing in one pass. Color, wetting solution, relative humidity and other parameters in the printing process are adjusted during the printing of all paper substrates. The paper is characterized by testing its surface properties, absorbency and mechanical properties. The results showed that a larger amount of paint, the side (front/back) of the print in uncoated and coated papers significantly affects the deviation in the register, also the deviation in the register is not the same in all positions of the sheets. Coated papers have higher resistance and breaking force compared to uncoated ones, but also a smaller contact angle with the wetting solution. By analyzing the correlation of deviations in the register and paper characteristics, their mutual connection was not observed. The experimental part of this work indicated the need for a wider range of paper substrates and the printing of a larger number of samples in order to obtain a statistically significant result.