Volatile organic compounds (VOCs) and odors in cattle rumen gas have been characterized by in-vivo headspace sampling by solid-phase microextraction (SPME) and analysis by gas chromatography–mass spectrometry–olfactometry (GC–MS–O). A novel device enabling headspace SPME (HS-SPME) sampling through a cannula was designed, refined, and used to collect rumen gas samples from steers. A Carboxen–polydimethylsiloxane (PDMS) fiber (85 μm) was used for SPME sampling. Fifty VOCs from ten chemical groups were identified in the rumen headspace. The VOCs identified had a wide range of molecular weight (MW) (34 to 184), boiling point (−63.3 to 292 °C), vapor pressure (1.05 × 10−5 to 1.17 × 102 Pa), and water solubility (0.66 to 1 × 106 mg L−1). Twenty-two of the compounds have a published odor detection thresholds (ODT) of less than 1 ppm. More than half of the compounds identified are reactive and have an estimated atmospheric lifetime of <24 h. The amounts of VFAs, sulfide compounds, phenolic compounds, and skatole, and the odor intensity of VFAs and sulfide compounds in the rumen gas were all higher after feeding than before feeding. These results indicate that rumen gases can be an important potential source of aerial emissions of reactive VOCs and odor. In-vivo sampling by SPME then GC–MS–O analysis can be a useful tool for qualitative characterization of rumen gases, digestion, and its relationship to odor and VOC formation.