This study aimed to screen the pathogenic genes and pathways that relate to the transformation of hamster buccal mucosa from precancerous lesions to squamous cell carcinoma by whole genome microarray and bioinformatics analysis. A DMBA (7,12-dimethylbenz[a]anthracene)-induced hamster model of a precancerous lesion and squamous cell carcinoma was established. The differentially expressed genes were detected using an Agilent whole rat genome microarray, which contains 41,000 genes/ESTs. Gene ontology (GO) functional classification and pathway analyses were performed, and a subset of differentially expressed genes were validated using RT-PCR. The results showed that during the transformation of hamster buccal mucosa from the precancerous lesion to squamous cell carcinoma, a total of 1,981 genes were differentially expressed, of which 1,037 were up-regulated and 944 were down-regulated. GO analysis revealed that the differentially expressed genes are mainly involved in 14 functional groups including those of metabolism and cell structure. Additionally, 9 significantly altered pathways were identified. Among the 1,861 known differentially expressed genes, 14 genes including Casp3, CCL5 and CXCL12 were enriched in the 9 altered pathways. The up-regulation of SPARC and down-regulation of Casp3 were confirmed by RT-PCR. In conclusion, a total of 1,981 differentially expressed genes and 9 significantly altered pathways were identified in the transformation of hamster buccal mucosa from precancerous lesions to squamous cell carcinoma. A total of 14 pathway-enriched genes including Casp3, CCL5 and CXCL12 may play critical roles in the alteration of cellular pathways leading to the transformation of buccal mucosa from precancerous lesions to squamous cell carcinoma. Future studies focusing on these genes and pathways are required in order to gain a better understanding and provide effective prevention and treatment of oral squamous cell carcinoma.