Cardiac hypertrophy (CH) is the main compensatory response to chronic heart stress and often progresses to a decompensation state potentially leading to heart failure. Phillyrin (PHI) is a novel compound derived from Forsythia, which has shown anti-inflammatory and anti-virus activities as well as renal protective effects on diabetic nephropathy. Therefore, we investigated the effects of PHI on CH induced by isoproterenol (ISO). Cardiac hypertrophy was induced by ISO in vivo, and the H9C2 cells were treated with ISO. PHI treatment alleviated CH in isoproterenol-induced mice in 7 and 14 days. Echocardiography showed that the PHI improved ISO-induced CH heart function and structure. PHI significantly decreased heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) ratios and improved left ventricular (LV) function in ISO-treated mice. Hematoxylin and eosin staining revealed cardiomyocyte areas of the ISO group were significantly increased, and PHI was significantly reduced at 7 and 14 days, PHI-100 groups showed significantly better improvements than PHI-50. Sirius red staining indicated PHI significantly decreased collagen deposition in heart cross-sections induced by ISO, and PHI repressed ISO-induced cTn-I and NT-proBNP expression in mouse serum. In vitro data from H9C2 cells showed that PHI decreased cell areas and total cell protein levels in cells induced by ISO, whereas ANP, BNP, IL-6, and IL-1β expression was significantly inhibited by PHI. Also, PHI simultaneously inhibited P65 and P38 phosphorylation in vivo and in vitro. In conclusion, this study demonstrated the protective effect of PHI on CH in in vivo and in vitro, and this effect was related to the suppression of inflammation through the activation of the P38/NF-κB pathway.