Chemotherapy upregulates inflammatory processes as measured by circulating concentrations of pro-inflammatory cytokines and their signaling lipids. Our objective was to observe if breast cancer chemotherapy influenced the circulating concentrations of provitamin A and non-provitamin A carotenoids and fat-soluble vitamins (FSVs) in free-living patients. Serum samples were collected from patients (n = 34) immediately prior to standard adjuvant and neo-adjuvant chemotherapy for breast cancer, and 4 months following chemotherapy commencement. Patient multivitamin and non-steroidal anti-inflammatory (NSAID) drug use was noted at both visits. Lipophilic extracts of serum were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to quantify α- and β-carotene, lycopene, lutein, zeaxanthin, β-cryptoxanthin, retinol, α-tocopherol and phylloquinone. Linear mixed models were developed to assess the relationship between the main factors (i.e., chemotherapy, multivitamin, and NSAID use), and their interaction effects, on serum carotenoid and fat-soluble vitamin concentrations. Random effects included a fixed intercept for each subject. Chemotherapy was significantly associated with reduced serum concentrations of α-carotene (P = 0.053) and retinol (P = 0.042), with a trend observed for reduced β-carotene (P = 0.076) and phylloquinone (P = 0.082). There was no main effect of multivitamin or NSAID use on any analytes investigated. An interaction effect was observed for chemotherapy * multivitamin use, with increased concentrations of serum retinol (P = 0.004) and lycopene (P = 0.004), and a trend observed for zeaxanthin (P = 0.087) for those who took multivitamins. Chemotherapy * NSAID use was also significantly associated with a trend in increased serum lutein (P = 0.061) for those who consumed NSAIDS. Our results suggest randomized, controlled trials of multivitamin use and/or provitamin A carotenoid-rich food consumption merit further investigation in patients undergoing chemotherapy treatment. This research was supported by The Ohio State University Stefanie Spielman Breast Cancer Center Kroger Fund, Pelotonia, NIH R01CA189947, NIH Award Number Grant P30 CA016058, OSU, and OSUCCC.