Fatigue is an intricate physiological phenomenon, causing serious secondary concerns and bioregulation disorders. The objective of this research was to look into the anti-fatigue effect and potential mechanisms of natural ingredients in methanol extract of edible oil from Rhus chinensis Mill. fruits (PMRO). Results showed that a total of 18 fatty acids and 9 anacardic acids were identified by GC-MS and UHPLC-ESI-HCES/MS in PMRO. PMRO significantly increased the weight-bearing swimming time. Further investigations showed that PMRO significantly improved physiological indexes related to fatigue and regulated the oxidative stress (Keap1/Nrf2) and energy metabolism (AMPK/PGC-1α)-related proteins expression to exert anti-fatigue effects. The 16S rRNA sequence analysis indicated that a potential role of PMRO in modulating the abundance of gut bacteria and influencing the short-chain fatty acids content, thereby exerting an anti-fatigue effect. Therefore, the natural ingredients in PMRO may hold promise as a dietary supplement capable of delaying body fatigue.