ALG3 has significant modulatory function in the process of tumor development. Yet how ALG3 involves in the advancement of different malignancies isn't fully understood. We performed a pan-cancer assessment on ALG3 utilizing datasets from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) to examine its tumor-related roles across malignancies and its link to particular molecules and cells in the tumor microenvironment (TME). Furthermore, we focused on breast cancer to examine the influence of ALG3-mediated signaling pathways and intercellular interactions in the advancement of tumors. The biological effects of ALG3 were verified by breast cancer cells. Enhanced ALG3 expression was discovered to be substantially linked to patients' grim prognoses in a number of malignancies. Furthermore, the expression of ALG3 in the TME was linked to the infiltration of stromal and immune cells, and ALG3-related immune checkpoints, TMB, and MSI were also discovered. We also discovered that cancer patients having a high level of ALG3 exhibited a lower probability of benefiting from immunotherapy. Furthermore, our research found that KEGG enrichment, single-cell RNA and spatial sequencing analyses were effective in identifying key signaling pathways in ALG3-associated tumor growth. In vitro, knockdown of ALG3 could decrease the proliferation of breast cancer cells. In summary, our research offers a comprehensive insight into the advancement of tumors under the mediation of ALG3. ALG3 appears to be intimately associated with tumor development in the TME. ALG3 might be a viable treatment target for cancer therapy, particularly in the case of breast cancer.