Objective of this study was to show the effect of different soil tillage systems onto humic substances content and quality. Furthermore chemical composition and structural differences in humic acids molecule in selected tillage systems were assessed. Long-term experiments were carried out on Luvi-haplic Chernozem (locality Unčovice, Olomouc region) during the period 2008-2011. Two types of tillage systems were studied: 1. plough till 0.35 – 0.40 m; 2. minimum loosening till 0.15 m. Humic acids were isolated according to the international standard method and characterized by elemental composition and 13C NMR spectroscopy. Spectrometer Varian INOVA 600 (frequency 150,830 MHz) was used. Results showed differences in quality of humic substances and higher content of fulvic acids in fractional composition under minimum tillage system. Analysis of chemical composition and structure of humic acids indicated higher content of aliphatic groups in HA molecule due to minimum loosening system. On the other hand humic acids isolated from ploughing soil were more aromatic. 13C NMR spectroscopy could be a useful tool to assess changes in structural composition of humic acids isolated from the different eco-systems