Animal welfare is a critical aspect of farming. Poor practices are strongly linked to deterioration in animal health and performance, as they suppress immune functions and stress. Negative handling can be a strong stressor while positive handling can reduce stress in farm animals. We hypothesized that handling stress would alter rumen environments and consequently microflora, which would reduce the ability of the rumen to digest hay. To test this hypothesis, we exposed four ewes to positive (free-grazing) and negative handling protocols (transportation or combination of novel stimulation and social isolation), as well as examined a control that received no treatment, and then compare the rumen liquid collected from all ewes. The extracted rumen liquid was subjected to in vitro grass hay degradability assay, and genetic approaches were used to identify ruminal microflora. It was shown that feed intake and in vitro grass hay degradability were reduced under the negative handling protocols. Further, sheep exposed to stress had more potentially pathogenic bacteria and fewer cellulolytic bacteria such as Firmicutes, as well as microorganism diversity in the rumen was lower. So, it was concluded that handling stress impairs the ability of the rumen to digest hay due to altered rumen microflora. It is therefore important to recognize potential animal production risks posed by handling stress, which has welfare implications for these farm animals.