This study was conducted to evaluate the viability of Lactobacillus casei strains and antioxidant action of bixin in probiotic butter as well as the acceptability of the product. Experimental samples of butter were divided into six formulations, involving two treatments: butter/bixin/isolate probiotic (IPBB); butter/bixin/commercial probiotic (CPBB) and four controls: butter/isolate probiotic (IPB); butter/commercial probiotic (CPB); butter/bixin (BB); and butter (B). In the IPBB and CPBB formulations, the L. casei count was ≥ 6 log CFU/g up to 74 and 69 days of storage at 4°C, respectively. The TBARS values, peroxide index, acidity, and short‐chain fatty acids showed an increase during storage for all formulations. The acceptability index was higher than 75%. The concentration of bixin did not affect the viability of the strains, nor did it influence the physicochemical aspects and acceptability of the product. Practical applications Fermented butter can be produced with the use of bixin (natural antioxidant), found in the seeds of annatto. Moreover, based on the regression models, it was found that the butter/bixin/isolate probiotic (IPBB) and butter/bixin/commercial probiotic (CPBB) formulations can be considered probiotic up to 74 and 69 days of storage, respectively, considering the concentration of ≥ 6 log CFU/g.