BACKGROUND: Single-cell RNA sequencing (scRNA-seq) enables the high-throughput profiling of gene expression at the single-cell level. However, overwhelming dropouts within data may obscure meaningful biological signals. Various imputation methods have recently been developed to address this problem. Therefore, it is important to perform a systematic evaluation of different imputation algorithms. RESULTS: In this study, we evaluated 11 of the most recent imputation methods on 12 real biological datasets from immunological studies and 4 simulated datasets. The performance of these methods was compared, based on numerical recovery, cell clustering and marker gene analysis. Most of the methods brought some benefits on numerical recovery. To some extent, the performance of imputation methods varied among protocols. In the cell clustering analysis, no method performed consistently well across all datasets. Some methods performed poorly on real datasets but excellent on simulated datasets. Surprisingly and importantly, some methods had a negative effect on cell clustering. In marker gene analysis, some methods identified potentially novel cell subsets. However, not all of the marker genes were successfully imputed in gene expression, suggesting that imputation challenges remain. CONCLUSIONS: In summary, different imputation methods showed different effects on different datasets, suggesting that imputation may have dataset specificity. Our study reveals the benefits and limitations of various imputation methods and provides a data-driven guidance for scRNA-seq data analysis.