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Abstract Background Technological advances have enabled transcriptome characterization of cell types at the single-cell level providing new biological insights. New methods that enable simple yet high-throughput single-cell expression profiling are highly desirable. Results Here we report a novel nanowell-based single-cell RNA sequencing system, ICELL8, which enables processing of thousands of cells per sample. The system employs a 5,184-nanowell-containing microchip to capture ~1,300 single cells and process them. Each nanowell contains preprinted oligonucleotides encoding poly-d(T), a unique well barcode, and a unique molecular identifier. The ICELL8 system uses imaging software to identify nanowells containing viable single cells and only wells with single cells are processed into sequencing libraries. Here, we report the performance and utility of ICELL8 using samples of increasing complexity from cultured cells to mouse solid tissue samples. Our assessment of the system to discriminate between mixed human and mouse cells showed that ICELL8 has a low cell multiplet rate (< 3%) and low cross-cell contamination. We characterized single-cell transcriptomes of more than a thousand cultured human and mouse cells as well as 468 mouse pancreatic islets cells. We were able to identify distinct cell types in pancreatic islets, including alpha, beta, delta and gamma cells. Conclusions Overall, ICELL8 provides efficient and cost-effective single-cell expression profiling of thousands of cells, allowing researchers to decipher single-cell transcriptomes within complex biological samples.