The relationship of weaning stress on fecal microbiota and serum and fecal metabolites of calm and temperamental 6-month-old Brahman bulls and heifers was studied. Temperament score (average of pen score and exit velocity) was used to classify calf temperament. Rectal fecal samples and jugular blood samples were collected from 10 calm and 10 temperamental calves (5 bulls and 5 heifers of each temperament) at weaning and d 4 after weaning. Fecal bacterial DNA was used to characterize microbiota by Illumina sequencing, MiSeq, QIIME, UniFrac, and analysis of similarities (ANOSIM) methods. Serum and fecal samples were used to characterize metabolites by untargeted GC-TOF mass spectrometry, ChromaTOF, and BinBase methods. Univariate analysis (alpha value for the Wilcoxon Mann-Whitney test = 0.05, FDR = 0.1) was used to determine differentially abundant microbial taxa and metabolites. Serum cortisol concentration was greater (P < 0.05) in temperamental than in calm calves. Serum cortisol was greater (P < 0.05) in heifers than bulls. The principal coordinate analysis (PCoA) plot showed 71 and 31 bacterial taxa were differentially enriched between day and sex at d 4, respectively; and 51 unknown and 9 known fecal metabolites were differentially enriched between day and sex at d 4, respectively. Abundance of metabolites of neuroactive compounds in fecal and serum samples varied by weaning, sex and temperament. Specifically, by d 4 5-aminovaleric acid, 5-methoxytryptamine, adenosine, capric acid, glutamic acid, tyramine, and 4-aminobutyric acid (GABA) were enriched (P < 0.05) in feces. By d 4 metabolites associated with lipolysis were differentially enriched in serum. Energy related metabolites (glycerol-3-galactoside, glucose and trehalose) were enriched (P < 0.05) in serum of temperamental relative to calm calves. Serum GABA was differentially enriched (P < 0.05) in heifers relative to bulls. The presence of microbiota taxa and metabolites associated with behavior and energy homeostasis were differentially affected by calf sex, temperament, and weaning stress.