PSY is a three-herb formula comprising Patriniae Radix, Mori Cortex Radicis, and Coix Seed that was previously shown to inhibit STAT3 signaling in colorectal cancer (CRC) cells. Here, we investigated molecular and metabolic responses to PSY, its antitumor activity in an HCT116 xenograft model, and its interactions with chemotherapy in vitro. Representative chemical markers were qualitatively verified by UPLC-ESI-QTOF MS/MS. Exploratory RNA sequencing with two biological replicates per group highlighted stress-associated and metabolic processes for further investigation. PSY increased BiP, phosphorylated eIF2α, ATF4, and CHOP levels, intracellular reactive oxygen species (ROS) levels, and mitochondrial membrane depolarization, while reducing the phosphorylation of PI3K/AKT/mTOR- and ERK-associated proteins. PSY increased the p-AMPK/AMPK ratio and decreased the p-ACC/ACC ratio. PSY reduced several lipid-biosynthetic transcripts and decreased the relative abundance of selected fatty acids in xenograft tumors. Oral administration of PSY suppressed HCT116 xenograft growth without significant body-weight loss. Combination-index (CI) analysis indicated CI-defined in vitro synergistic interactions with 5-fluorouracil (5-FU) at multiple concentration pairs, with interaction patterns varying across HCT116, HT-29, and SW480 cells. Direct cell counting supported the combination response in HCT116 and SW480 cells. The combination increased apoptosis and reduced STAT3 and NF-κB p65 phosphorylation relative to single treatments. These findings characterize concurrent, partly discordant signaling and metabolic responses to PSY and support further preclinical evaluation of its in vitro interaction with 5-FU.