Abstract The seaweed Gracilariopsis lemaneiformis is widely cultivated in the southern and northern coasts of China, and is mainly used for agar extraction and abalone feed. The cultivated strain of G. lemaneiformis 981 has various advantages over the wild type such as growth rate and high-temperature tolerance. Therefore, it is necessary to investigate the differences between the wild-type and cultivated G. lemaneiformis. Here, 648 up-regulated and 837 down-regulated proteins were screened using the isobaric tags for relative and absolute quantitation (iTRAQ) technique, and these differentially expressed proteins (DEPs) were concentrated in the pathways including “Ribosome”, “Photosynthesis-antenna proteins”, “alpha-Linolenic acid metabolism”, and “Phenylpropanoid biosynthesis”. The most prominent pathway was “Ribosome”, namely, 116 out of 119 ribosome-associated proteins were up-regulated in G. lemaneiformis 981 compared to the wild type. Subsequently, multiple reaction monitoring (MRM) validated the credibility of the iTRAQ results. Finally, “Phenylpropanoid biosynthesis” and “Photosynthesis-antenna proteins” pathways were also found to be significantly changed in G. lemaneiformis 981 validated by gene expression profiling analysis. Altogether, these results, in combination with chlorophyll fluorescence parameters, unveiled the possible mechanism of differences in growth and anti-abiotic stresses between the wild-type and cultivated G. lemaneiformis, which would provide a reference for breeding of excellent seaweeds.