*It is generally hypothesized that the synthesis of cellulose in higher plants is mediated by cellulose synthase complexes (CSCs) localized on the plasma membrane. However, CSCs have not been investigated thoroughly through their isolation. The availability of ample Populus tissue allowed Populus CSCs to be isolated and characterized in association with xylem differentiation. *The methods used here included co-immunoprecipitation, proteomic analysis, laser microdissection, immunolocalization and others. *Western blot analysis of the immunoprecipitated CSCs led to the identification of at least two types of CSC in the membrane protein of Populus xylem tissue. Proteomic analysis further revealed that the two types of CSC were assembled from different cellulose synthase proteins. Immunolocalization confirmed that both types of CSC were involved in secondary cell wall formation. In addition, a number of noncellulose synthase proteins were also identified in association with CSC precipitation. *The results indicate that two types of CSC participate in secondary wall formation in Populus, suggesting a new mechanism of cellulose formation involved in the thickening of wood cell walls. This study also suggests that the CSC machinery may be aided by other proteins in addition to cellulose synthase proteins.