HL- sp1 , a 6.7 kb HL-CMS-related fragment, was cloned from the mitochondrial genome of HL-CMS sterile line YTA. One of the two ORFs predicted encodes 290 amino acids and is tentatively named orf290 . One overexpression vector 35S∶∶Rf1b5′∶∶orf290 with a mitochondrial signal peptide was constructed and transformed by Agrobacterium into YTB, a HL-CMS maintain line. The copy numbers of the exogenous gene in the 35S∶∶Rf1b5′∶∶orf290 transgenic T0 plants were estimated according the ratio of hygromycin B phosphotransferase ( hpt ) gene and the single copy sucrose phosphate synthase ( SPS ) gene in rice with qPCR based on DNA. The pollen fertility and the seed set rate of the T0 positive transgenic plants were investigated. The results showed that two transgenic plants with single copy of 35S∶∶Rf1b5′ ′∶∶ orf290 were obtained and the expression level of orf290 in the T0 positive transgenic plants were detected with qPCR. The average pollen fertility was 30.8% and the average seed set rate was 41.6% in the single copy transgenic lines. It is indicated that mitochondrial gene orf290 may be related to cytoplasmic male sterility in rice.