An active compound of citrus oils (CO), citral, contributes to the bactericidal activity of CO and serves as a non-antibiotic alternative candidate for the treatment of bovine Escherichia coli mastitis. During times of inflammation, monitoring the immune response can generate economic and welfare benefits due to the high incidence of mastitis across the dairy industry. The objective of this study was to evaluate the immune response after being challenged with mastitis pathogen E. coli strain P4. Eighteen healthy, multiparous Holstein cows in mid-lactation (>100 d in milk) were challenged with 800 cfu of E. coli strain P4 into one rear mammary quarter. One of 3 intramammary treatments were administered into the infected quarter ~24 h post-infection including: 1) CON: sterile phosphate buffer solution twice daily, 2) CO: 1.0% (vol/vol) citral twice daily or 3) AB: ceftiofur hydrochloride once daily (based on label instructions). Coccygeal blood was collected daily through d 7 post-challenge where serum was harvested. Serum haptoglobin was analyzed using a commercially available kit (Tridelta Development Limited, Dublin, Ireland). Data were analyzed using ANOVA of the PROC MIXED procedure of SAS v.9.4. The results indicated that serum haptoglobin was not different by treatment (P = 0.55) or for the interaction of treatment by day of challenge (P = 0.97). In conclusion, CO does not alter serum haptoglobin response even when compared with the control during E. coli bovine mastitis and CO may not serve as an alternative treatment for bovine mastitis infection with a gram-negative organism.