The guaianolide family of sesquiterpene lactones is known for its distinctive structural features and diverse biological activities. 4,9,10-Trihydroxyguaia-11(13)-en-12,6-olide, with an underdetermined absolute stereochemistry ( or -), is a newly identified 6,12-guaianolide isolated from the genus . Motivated by the potential biological activity of the natural product, we pursue its stereoselective synthesis. Starting from ()-limonene, an asymmetric total synthesis of 4α,9α,10α-trihydroxyguaia-11(13)-en-12,6α-olide () is accomplished in 20 steps with an overall yield of 4%, utilizing key transformations such as stereoselective reductive epoxide opening and additions of methyl lithiopropiolate and allyl cuprate. Most significantly, preliminary biological testing uncovers new anticancer activity of against rare and aggressive childhood atypical teratoid rhabdoid tumor (ATRT) and other cancer cell lines. We anticipate that our synthetic strategy will enable the development of chemical probes and derivatives derived from for mechanism of action studies and anticancer drug discovery.