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In order to decipher the hygromycin B tolerance and resistance mechanisms of Nannochloropsis oceanica, the transcrip-tome profiles of a transgenic strain carrying a randomly integrated hygromycin B resistant gene, a hygromycin B-adaptive strain and a wild type strain of N. oceanica were compared by transcriptome sequencing (RNA-seq) without referring to a high quality genome sequence. The results showed that the adaptive strain adapts to the hygromycin B existing environments mainly by intensifying the expressions of the efflux pump ABC and MFS superfamily transporter genes, thus reducing the intracellular concentration of hygro-mycin B. The transgenic strain obtains the hygromycin B resistance ability solely by expressing exogenous resistance gene. Accord-ingly, the screening and maintenance of N. oceanica transformants should be carried out at an antibiotics concentration higher than the adaptive threshold. Our findings can help the genetic modification of N. oceanica with the application of hygromycin B.