Background: Aberrant glycosylation is a hallmark of cancer and thereby has an excellent potential for the discovery of novel biomarkers. Impairments in the glycan composition of lipoproteins impact their functional properties and can be associated with various diseases, including cancer. This research is still in its infancy; however, it can lead to the development of new diagnostic and disease stratification approaches as well as therapeutic strategies. Therefore, we aimed to evaluate anomalies in O-glycosylation of apolipoprotein C-III (apoC-III) in colorectal carcinoma (CRC) patients’ sera, in comparison with sera from healthy individuals, and assess the disparities of O-glycoforms on apoC-III in CRC. Methods: The choice of patients (n = 42) was based on the same tumor type (adenocarcinoma) and tumor size (T3), without or with inconsiderable lymph node infiltration. Patients with comorbidities were excluded from the study. The control healthy individuals (n = 40) were age- and sex-matched with patients. We used an approach based on the MALDI-TOF MS in linear positive ion mode, allowing simple analysis of O-glycosylation on intact apoC-III molecules in the serum samples directly, without the need for specific protein isolation. This approach enables relatively simple and high-throughput analysis. Results: In CRC patients’ sera samples, we observed significantly elevated apoC-III sialylation. Fully sialylated (disialylated) O-glycans had 1.26 times higher relative abundance in CRC samples compared to controls with a p-value of Mann–Whitney U test of 0.0021. Conclusions: We found altered O-glycosylation of apoC-III in the serum of CRC patients. However, it can be non-specific as it may be associated with another process such as ongoing inflammation. Therefore, to establish it as a potential novel non-invasive biomarker for CRC in suspected patients, further studies interrogating the changes in apoC-III O-glycosylation and the robustness of this biomarker need to be performed and evaluated.