Abstract Background Lysine acetylation is a post-translational modification that regulates a diversity of biological processes, including cancer development. Methods Here, we performed the quantitative acetylproteomic analysis of three primary cervical cancer tissues and corresponding adjacent normal tissues by using the label-free proteomics approach. Results We identified a total of 928 lysine acetylation sites from 1547 proteins, in which 495 lysine acetylation sites corresponding to 296 proteins were quantified. Further, 41 differentially expressed lysine acetylation sites corresponding to 30 proteins were obtained in cervical cancer tissues compared with adjacent normal tissues (Fold change > 2 and P < 0.05), of which 1 was downregulated, 40 were upregulated. Moreover, 75 lysine acetylation sites corresponding to 58 proteins were specifically detected in cancer tissues or normal adjacent tissues. Motif-X analysis showed that kxxxkxxxk, GkL, AxxEk, kLxE, and kkxxxk are the most enriched motifs with over four-fold increases when compared with the background matches. KEGG analysis showed that proteins identified from differently and specifically expressed peptides may influence key pathways, such as Notch signaling pathway, viral carcinogenesis, RNA transport, and Jak-STAT, which play an important role in tumor progression. Furthermore, the acetylated levels of CREBBP and S100A9 in cervical cancer tissues were confirmed by immunoprecipitation (IP) and Western blot analysis. Conclusions Taken together, our data provide novel insights into the role of protein lysine acetylation in cervical carcinogenesis.