Introduction Breast cancer (BC) is still the most common malignancy among women globally, and triple-negative breast cancer (TNBC) presents major therapeutic and management issues due to its aggressive nature. Recent studies suggest that the vitamin D receptor (VDR) and estrogen receptor beta 1 (ERβ1) play crucial roles in regulating TNBC progression. Increased expression of VDR and ERβ1 has been linked to tumor suppression, highlighting their potential to impact cancer progression via various signaling pathways. This study analyzes VDR and ERβ1 expressions in TNBC subtypes to discover potential therapeutic targets and improve treatment outcomes for this challenging BC subtype. Method This cross-sectional study analyzed 30 invasive ductal carcinoma (IDC) cases of TNBC subtypes using formalin-fixed paraffin embedding (FFPE) tissues. Immunohistochemistry assessed cytoplasmic and nuclear VDR and ERβ1 expression, scoring staining intensity and extent, categorized as negative/low, moderate, or high expression. Results High VDR and ERβ1 expressions were analyzed across molecular subtypes of TNBC to explore their therapeutic potential, particularly in TNBC. In TNBC, a high VDR expression was observed in the cytoplasm (n = 10, 33.3%) and the nucleus (n = 2, 6.6%), with statistical significance (p <
0.042). Luminal A cases demonstrated high VDR expression in the cytoplasm (n = 6, 20%) and the nucleus (n = 2, 6.6%) (p <
0.042), while luminal B exhibited high VDR expression exclusively in the cytoplasm (n = 4, 13.3%) (p <
0.042). In HER2-enriched, high VDR expression was confined to the nucleus (n = 3, 10%) (p <
0.042). ERβ1 expression patterns in TNBC showed moderate cytoplasmic expression (n = 9, 50%) and high cytoplasmic expression (n = 1, 5.5%), with statistical significance (p <
0.025). By contrast, luminal A displayed moderate cytoplasmic expression (n = 3, 16.6%) and high cytoplasmic expression (n = 5, 27.7%) (p <
0.025). These findings suggest that VDR and ERβ1 exhibit subtype-specific expression patterns, with significant expression in TNBC, indicating their potential as therapeutic targets. Conclusion VDR and ERβ1 expressions differ between TNBC subtypes, indicating their potential as targeted therapies, particularly in TNBC.