Land application of swine lagoon effluent (SLE) is widespread in southeastern USA and often results in changes in soil properties.  Because soil enzymes are highly involved in the degradation of soil organic matter and nutrient cycling, a better assessment on the relationship of soil enzyme activities with SLE application may facilitate informed management decisions.  We evaluated the activities of a suite of soil enzymes important to soil C, N and P cycling as a consequence of 3-y consecutive application of SLE versus ammonium nitrate (AN) at a rate equivalent to 0, 200, 400 or 600 kg plant available N ha^-1 y^-1.  Enzyme activities varied differently for soils applied with SLE versus AN.  While the activities of enzymes involved in N and P cycling were very sensitive to reduction in soil pH due to SLE or AN application at a high rate, the activities of enzymes involved in C cycles varied significantly with the source of fertilization.  Compared to the unfertilized control, the activities of oxidative enzymes (i.e. phenol oxidase and peroxidase) reduced or remained as a consequence of 3-y consecutive AN fertilization.  However, those enzyme activities were stimulated by the SLE application.  The differences in soil enzyme activities between SLE- and AN-fertilized soils were presumably due to the changes in soil microbial community compositions, resulting partially from long-lasting changes in soil pH.  Non-metric multidimensional scaling analysis further showed that soil enzyme activities differed significantly (P < 0.05) between fertilized soil and unfertilized control and between SLE- and AN-fertilized soils. These differences were significantly correlated to soil pH (Pearson’s correlation coefficient r = 0.76, P < 0.05), indicating soil pH was a critical factor regulating soil enzyme activities.