This work characterized ruminal microbial gene expression shifts during heat stress or intake restriction, and quantitatively related these shifts to changes in volatile fatty acid (VFA) production, absorption, and interconversion. Non-steady-state, ¹³C-labeled VFA were used to determine effects of heat stress or intake restriction on VFA metabolism in 8, 6 mo old Holstein heifers. A 2 × 2 factorial design with 2 periods and 4 groups was used. In period 1, both groups had ad libitum access to feed and were housed at 20°C. In period 2, temperature was increased to 30°C in one group (HS; n = 4) and the other group (n = 4; 20°C) was pair-fed so that intake matched HS. Analysis of isotope ratios and concentrations of VFA using a fully exchanging, dynamic 3-pool model revealed significant (P < 0.05) shifts in VFA fluxes associated with heat stress and intake restriction. RNA isolated from rumen fluid and solid in each period was sequenced using the Illumina HiSeq (2×100 base pairs) platform. After removing short and poor quality reads with SortMeRNA, and screening out host genes using TopHat, MetaVelvet was used for de novo genome assembly. Contigs were annotated using USEARCH against the CAZy database and read-corrected gene abundance was analyzed using HUMAnN2. Gene abundances for microbial enzymes were regressed on VFA fluxes to determine quantitative relationships. Production rates of VFA were significantly (P < 0.05) related to microbial gene expression of enzymes involved in the breakdown of glycosidic bonds suggesting fiber and starch degradation are key steps in VFA production. Interchange among VFA was significantly (P < 0.05) related to glycogen degrading enzymes, suggesting a portion of C transfer among VFA occurs through microbial glycogen or that conditions promoting glycogen storage also promote interconversions. Variation in microbial enzyme gene expression explained 67 to 94% of the variation in VFA production, 74 to 98% of variation in interconversions, and 69 to 99% of variation in absorption, indicating high potential to leverage microbial gene expression patterns to enhance understanding of factors regulating VFA metabolism.