Physalis minima is a medicinal and edible plant in China. In this study, 22 new withaphysalins, including a novel 1(10 → 6)abeo-14β-hydroxy one (1) and other 15 unusual 14β-hydroxy ones (3-4, 6-17, 19), were isolated from the whole herbs of P. minima together with two known analogues (23-24). Their structures were established by extensive analysis of high-resolution electrospray ionization mass spectrometry, IR, and 1D and 2D NMR spectroscopic data. Their absolute configurations were determined by electronic circular dichroism (ECD) spectra and single-crystal X-ray crystallographic analyses, together with DFT NMR calculations. All isolated compounds were evaluated for their anti-inflammatory activity via measuring the colorimetric reporter of the secreted embryonic alkaline phosphatase gene driven by an IFN-β minimal promoter fused to five copies of the NF-κB consensus transcriptional response element and three copies of the c-Rel binding site in LPS-stimulated human THP1-Dual cells. Compounds 2, 5, 6, 9, 10, 11, and 20 showed significant anti-inflammatory effects with IC50 values in the range of 3.01-13.39 μM. Among them, compounds 2 and 10 showed better anti-inflammatory effects to inhibit the secretion of IL-6, IL-1β, and TNF-α in LPS-stimulated THP1-Dual cells.