Influence of ionic strength (I) on the pyrene binding constant (Koc) in six different humic substance (HS) solutions, three soil humic acids, one freshwater humic acid, one freshwater fulvic acid, and one freshwater natural organic matter was investigated by fluorescence quenching technique. Specific two-stage changes of Koc with increasing ionic strength were only observed in three soil humic acid solutions with varying to different extents. Two primary governing factors for the differences of these HS systems are binding potential and the abundance of acidic functional groups (carboxyl and phenolic) of HS. Experimental results demonstrated that within the ionic strength range studied in the present research, ionic strength had a major effect on molecular size of HS and brought about change of Koc. The salting-out effect did not. The change in molecular size was caused by the associations between cations and acidic groups of HS. Using pyrene as a probe, we also investigated the interaction of cation and dissolved HS and found relatively slow kinetics (at least 5 d), leading us to believe that the special cation-HS kinetics may influence the results of geochemical studies involving the measurement of ionic strength effect.