Natural antisense transcripts (NATs) constitute a class of non-coding RNAs that have emerged as important regulators of gene expression. However, involvement of NATs in colorectal cancer (CRC) development has not been reported to date. In the present study, the up- and down-regulation of NATs were investigated in human CRC for their possible involvement in CRC development. Total RNAs isolated from 51 CRC tissues, 9 corresponding non-cancerous tissues and 19 liver metastatic tissues from surgically resected samples were subjected to expression analysis using a custom-microarray containing human sense/antisense probes for ca. 21,000 genes. Comparing CRC tissues with non-cancerous tissues, we identified 415 NATs differentially expressed in CRC and non-cancerous tissues to a significant degree (p<0.001, fold change >4.0 or ≤4.0). When a hierarchical clustering was performed on CRC and non-cancerous samples using these 415 NATs, the samples were separately clustered. Principal component analysis with the same NATs showed clear separation of CRC and non-cancerous samples using the first two principal components (PC1, 80%; PC2, 10%). To validate the expression results obtained from the microarray, the expressions of the 3 selected NATs were examined by strand-specific RT-qPCR, revealing that these expression profiles were consistent with those obtained from microarray analysis. In addition, the NAT expression patterns were found to be different between primary tumors with liver metastasis and those without liver metastasis. In conclusion, these findings taken together indicated that NATs identified in the present study would be involved in CRC development as well as possibly in its metastasis.