To investigate the effect of 5-aminolevulinic acid (5-ALA) on in vitro rumen gas production, fermentation characteristics, and bacterial community profiles, five levels of 5-ALA (0, 100, 500, 1000, and 5000 mg/kg DM) were supplemented into a total mixed ration (concentrate/forage = 40:60) as substrate in an in vitro experiment. Results showed that as the supplementation level of 5-ALA increased, asymptotic gas production (b) decreased linearly and quadratically ( < 0.01) while the dry matter degradation rate increased quadratically ( < 0.01). Meanwhile, the propionate concentration of 72 h incubation fluid increased linearly ( = 0.03) and pH value increased linearly and quadratically ( < 0.01), while the concentrations of butyrate, isobutyrate, valerate, isovalerate, and NH-N and the ratio of acetate/propionate (A/P) decreased linearly and quadratically ( < 0.05). There was no significant difference in any alpha diversity indices of bacterial communities among the various 5-ALA levels ( < 0.05). PCoA and PERMANOVA analysis revealed that the bacterial profiles showed a statistical difference between the treatment 5-ALA at 1000 mg/kg DM and the other levels except for 5000 mg/kg DM ( < 0.05). Taxonomic classification revealed a total of 18 and 173 bacterial taxa at the phylum and genus level with relative abundances higher than 0.01% in at least half of the samples, respectively. LEfse analysis revealed that 19 bacterial taxa were affected by 5-ALA levels. Correlation analysis showed that was positively correlated with the gas production parameter b, the ratio of A/P, and the concentration of butyrate, isovalerate, and NH-N ( < 0.05) and negatively correlated with pH ( < 0.05). exhibited a negative correlation with the gas production parameter b, the ratio of A/P, and the concentration of butyrate, valerate, isobutyrate, isovalerate, and NH-N ( < 0.05), along with a weaker positive correlation with pH ( = 0.04). The was negatively correlated with the concentration of propionate but positively correlated with gas production parameter b and the concentration of butyrate and NH-N ( < 0.05). The was found to have a positive correlation with gas production parameter b, the ratio of A/P, and the concentration of butyrate, isobutyrate, isovalerate, valerate, total VFA, and NH-N ( < 0.05), but a highly negative correlation with pH ( < 0.01). Differential metabolic pathways analysis suggested that metabolic pathways related to crude protein utilization, such as L-glutamate degradation VIII (to propanoate), L-tryptophan degradation IX, and urea cycle, increased with 5-ALA levels. In summary, including 5-ALA in the diet might improve energy and protein utilization by reducing the abundance of , the , the , and certain pathogenic bacteria and increasing the abundance of WPS-2.