Plant-parasitic cyst nematodes secrete cell wall modifying proteins during their invasion of host plants. In this study, we used a monoclonal antibody to immunopurify and to sequence the N terminus of the most abundant cellulases in stylet secretions of preparasitic juveniles of Globodera rostochiensis. The N-terminal amino acid sequence perfectly matched the sequence of an expressed sequence tag of two nearly identical genes, named Gr-eng3 and Gr-eng4, which show relatively low similarity with the previously identified Gr-eng1 and Gr-eng2 (i.e., 62% similarity and 42% identity). The recombinantly produced proteins from Gr-eng3 and Gr-eng4 demonstrated specific activity on carboxymethylcellulose, indicating that these genes encode active cellulases. To date, the cellulases in cyst nematodes are comprised of three possible domain structure variants with different types of ancillary domains at the C terminus of the glycosyl hydrolase family 5 (GHF5) domain. We used Bayesian inference to show that the phylogeny of the GHF5 domain based on currently available data suggest that the extant nematode cellulases arose through reshuffling of the GHF5 domain with different types of ancillary domains as relatively independent units. Knocking-down Gr-eng3 and Gr-eng4 using RNA interference resulted in a reduction of nematode infectivity by 57%. Our observations show that the reduced infectivity of the nematodes can be attributed to poor penetration of the host's root system at the onset of parasitism.