Background: Ectopic pregnancy (EP), characterized by the implantation of a fertilized ovum outside the uterine cavity, remains a major cause of maternal morbidity and mortality in early pregnancy. Despite advances in imaging and hormonal diagnostics, early and accurate identification of EP remains a clinical challenge. Emerging evidence suggests that cellular stress responses, particularly endoplasmic reticulum (ER) stress, and metabolic disturbances caused by gut microbiota-derived metabolites such as trimethylamine N-oxide (TMAO) may contribute to impaired trophoblast function and abnormal implantation. However, the relationship between ER stress signaling pathways and TMAO in the context of EP has not yet been elucidated. Understanding these mechanisms could pave the way for the development of new biomarkers to improve early diagnosis and risk stratification in EP. Objective: The aim of this study was to investigate the role of ER stress markers and TMAO, a metabolite from the gut microbiota, in EP. Study design: A prospective study was conducted with 88 pregnant women, including 44 with EP and 44 with healthy intrauterine pregnancies, matched for age and weeks of gestation. Serum levels of ER stress markers (GRP78, IRE1, PERK, SREBP-1c, XBP1) and TMAO were measured using ELISA and LC–MS/MS methods. Hematologic parameters were also compared between the groups. Correlations between TMAO and ER markers were analyzed, and diagnostic performance was evaluated by ROC analysis. Results: The levels of GRP78, IRE1, PERK, SREBP-1c, XBP1 and TMAO were significantly increased in the EP group compared to the controls (all p < 0.001). Positive correlations were observed between TMAO and PERK (ρ = 0.571) and SREBP-1c (ρ = 0.647). Among hematologic findings, WBC and platelet counts were significantly higher, while HGB, MCH and MCHC levels were significantly lower in EP patients. ROC analyses showed that SREBP-1c (AUC = 0.999), PERK (AUC = 0.935) and TMAO (AUC = 0.932) had excellent diagnostic performance. Conclusion: The results suggest that both ER stress and microbiota-induced metabolic dysfunction play a role in the pathophysiology of EP. Elevated serum levels of SREBP-1c, PERK and TMAO could serve as promising diagnostic biomarkers for the early detection of EP. Further studies with larger and multicenter samples are warranted to validate these findings and investigate their clinical applicability.