Histone deacetylase (HDAC) inhibition attenuates inflammation in rodents and short-chain fatty acids (SCFAs) are effective HDAC inhibitors. Therefore, the objective of this study was to evaluate the role of the SCFAs sodium propionate (SP) and sodium butyrate (SB) as HDAC-dependent regulators of inflammatory gene expression in bovine mammary epithelial cells (MAC-Ts). We postulated that SP and SB would decrease inflammation in MAC-Ts by inhibiting HDAC activity and increasing histone H3 acetylation and consequently decreasing inflammatory gene expression. For this study, MAC-Ts stimulated with lipopolysaccharide (LPS) were used as a model for bovine mammary epithelial cell inflammation. MAC-Ts were cultured in a basal medium. Cell lysates were incubated with SP or SB (0 to 5 mM) for 2 h prior to HDAC substrates incubation for an additional 2 h and HDACs activity was determined. Next, cells were pretreated with SP or SB (0 to 3.0 mM) for 2 h prior to LPS (1 µg/mL) stimulation for an additional 2 h and assessed for histone H3 acetylation. Then, cells were pretreated with SP or SB (1 mM) for 24 h prior to LPS (1 µg/mL) stimulation for an additional 2 h and RNA was isolated for inflammatory gene expression evaluation by PCR array and gene validation was performed using quantitative real-time PCR. One-way ANOVA followed by Tukey post hoc analysis was conducted and statistical significance set at P < 0.05. SP and SB concentration-dependently and selectively inhibited class I HDAC activity, which differed between SCFAs, where SB inhibited (P < 0.05) HDACs 2, 3, and 8, while SP inhibited (P < 0.05) HDACs 2 and 8. Histone H3 acetylation was concentration-dependently increased by SCFAs and likewise the differential regulation of HDAC activity, SCFAs effected differently histone H3 acetylation, where SB increased (P < 0.05) H3K9/14, H3K18 and H3K27 acetylation, while SP increased (P < 0.05) H3K9/14 and H3K18 acetylation. However, SCFAs did not decrease (P > 0.05) overall inflammatory gene expression. Under our experimental conditions, findings suggest that in MAC-Ts, SCFAs regulate epigenetic markers on nucleosomal DNA in addition to regulation of inflammatory gene events independent of HDAC activity. Nevertheless, examination of SCFAs and/or HDACs inhibitors in bovine mammary gland is worth being further investigated to delineate the potential impact of HDAC inhibition and histones hyperacetylation on mammary gland tissue inflammation.