Abstract: The objective of present study was to construct a subtractive cDNA library for infective larvae of Ascaris suum, for the identification and cloning of differentially expressed genes in infective larvae of A. suum. The subtractive cDNA library for infective larvae of A. suum was constructed with the technique of suppression subtractive hybridization (SSH) using the PCR-Select cDNA Subtraction kit (Clontech) after subtraction with cDNAs representing other developmental stages (adults, eggs, L3 and L4). The specificity of the constructed archives was determined using Southern dot blot, followed by sequencing of 45 random clones showing significant expression in the infective stage. The results showed that the subtractive cDNA library for infective larvae of A. suum was specific highly as confirmed by Southern dot blot. 40 of the 41 valid expression sequence targets (ESTs) had significant similarities with that of other known genes, mainly representing L3 genes and female head gene of A. suum. One EST may represent a new gene. The successful construction of the subtractive cDNA library for infective larvae of A. suum provides a foundation for further studies of genes related to the infectivity and migration of A. suum larvae.