Ubiquitination is an important post-translational modification that mediates protein degradation in eukaryotic cells participating in multiple biological processes. However the profiling of protein ubiquitination and the function of this crucial modification in fruit ripening remain largely unknown. In this study we found that suppression of proteasome by the inhibitor MG132 retarded strawberry fruit ripening. Using K-epsilon-GG antibody enrichment combined with high-resolution mass spectrometry we performed a comprehensive ubiquitinome analysis in strawberry fruit. We identified 2947 ubiquitination sites for 2878 peptides within 1487 proteins which are involved in a variety of cellular functions. The lysine at position 48 (K48)-linked poly-ubiquitin chains appeared to be the most prevalent type of modification among the identified ubiquitinated proteins. A large number of ubiquitination sites exhibited altered ubiquitination levels after proteasome inhibition including those within ripening-related proteins associated with sugar and acid metabolism cell wall metabolism anthocyanin synthesis and ABA biosynthesis and signalling. We further demonstrated that FaBRIZ a RING-type E3 ligase functions as a negative regulator of ripening in strawberry fruit. Our findings highlight the critical regulatory roles of protein ubiquitination in fruit ripening. The ubiquitinome data provide a basis for further exploration of the function of ubiquitination on specific proteins.