Acidification of freshwaters via anthropogenic means is a global problem. Decreases in pH in these systems have devastating effects on macroinvertebrate fauna especially Ephemeropterans. However, fauna in naturally acidic waters with high concentrations of humic substances (HS) appear to be less affected by acidification. This study investigated the ability of HS to decrease toxicity of low pH to mayflies (Atalophlebia spp.) in both hard and soft water environments. Laboratory trialling with both hard and soft water was conducted to represent water chemistries commonly associated with anthropogenic and naturally acidic freshwaters. Mayflies were exposed to decreases in pH between 7 (control) and 3.5 in the presence of 0, 10 and 20mg/L HS (Aldrich humic acid). HS increased mayfly survival by up to 45% in soft water and up to 39% in hard water. HS were also shown to increase 96h LC50 values in both soft (4.29, 0mg/L; 3.99, 10mg/L and 3.97, 20mg/L) and hard water (4.58, 0mg/L; 4.22, 10mg/L and 4.07, 20mg/L). The results of this study are important in showing that HS can influence the toxicity of low pH environments; thus providing insight into why contrasting effects on biota are recorded from naturally acidic compared with anthropogenically-acidified environments.