FUS3 transcription factor is a key regulator of seed oil synthesis in plants. In order to investigate the role of the CsFUS3 gene in lipid synthesis and accumulation of Camelina sativa, we conducted a genomewide identification of the CsFUS3 gene family, analyzed the spatiotemporal expression patterns of CsFUS3 genes, and analyzed the function of CsFUS31 and CsFUS32 genes in lipid synthesis. This study provides a theoretical foundation for indepth analysis of the function of CsFUS3 genes in the oil synthesis and the breeding of high oil varieties of C. sativa. The results showed: (1) using the AtFUS3 protein sequence, two complete CsFUS3 protein sequences were identified from the C. sativa genome database, named CsFUS31 and CsFUS32, respectively. The subcellular localization result showed that the two CsFUS3 proteins were located in the nucleus. (2) CsFUS31 and CsFUS32 proteins of C. sativa were most closely related to Arabidopsis AtFUS3 protein. They had similar physicochemical properties, advanced structure, and complete B3 functional domain to Arabidopsis AtFUS3 protein. (3) The results of qRTPCR showed that the CsFUS31 and CsFUS32 genes were only expressed in seeds, and with the seeds matured, the expression levels of the two CsFUS3 genes showed a trend of increasing at first and then decreasing, and the expression level reached the highest level at 30 days after flowering. (4) The interaction of CsFUS3 and CsWRI1 proteins and the transcriptional regulation of OLE and ABI3 genes by CsFUS3 may be key regulatory pathways of high oil traits in C. sativa. (5) The transient expression analysis of tobacco showed that compared with the wild type, the total oil content of tobacco leaves transfected with CsFUS31 and CsFUS32 genes increased by 0.95% and 1.12%, respectively. It indicated that the CsFUS3 genes could increase the synthesis and accumulation of total oil in tobacco leaves.