Enteric methane (CH₄) emissions from ruminants are one of the major contributors to greenhouse gas emissions (GHG) from agriculture. We hypothesized that increasing levels of whole cottonseed (WCS) in diets decreases CH₄ production in ruminants fed low-quality forages without compromising feed digestibility. Dietary lipid supplementation, particularly with oilseeds such as WCS, has the potential to reduce enteric CH₄ production by altering rumen fermentation pathways. This study aimed to evaluate the effects of WCS inclusion in a tropical forage-based diet on in vitro gas and CH₄ production, fermentation characteristics, and in vitro dry matter digestibility (IVDMD). Five treatments were tested using guinea grass hay (GG) supplemented with 0, 12.5, 25, 37.5, and 50% of WCS (DM basis). Results after 48 h of incubation showed a quadratic response of total gas production with increasing WCS inclusion (p < 0.01), with the highest values observed at 12.5% and 25% WCS. Methane production from digested DM significantly reduced at 50% WCS inclusion (−10.66% respect to control; p = 0.02), suggesting a mitigation effect without detrimental impacts on IVDMD (p = 0.16), which remained unaffected across all treatments. No effects were found in initial or final pH (p = 0.98 and p = 0.89, respectively) or total protozoa count among treatments (p = 0.99). However, levels of 50% WCS inclusion exceed the recommended limit of fat in ruminant diets (>6% DM). Further in vivo validation trials are recommended to confirm these in vitro results and evaluate long-term impacts on animal performance and CH₄ emissions. This article is a revised and expanded version of Whole cottonseed as an alternative to mitigate in vitro methane emissions on low-quality forage-based diets. Presented at the International Research Symposium on Agricultural Greenhouse Gas Mitigation. From Research to Implementation. 21–24 October 2024, Berlin, Germany.