Cathelicidin hCAP18/LL-37 can resist infection from various pathogens and is an essential component of the human immune system. Accumulating evidence has indicated that hCAP18/LL-37 plays a tissue-specific role in human cancer. However, its function in hepatocellular carcinoma (HCC) is poorly understood. The present study investigated the effects of hCAP18/LL-37 on HCC in vitro and in vivo. Results showed that hCAP18/LL-37 overexpression significantly promoted the proliferation of cultured HCC cells and the growth of PLC/PRF-5 xenograft tumor. Transcriptome sequencing analyses revealed that the PI3K/Akt pathway was the most significant upregulated pathway induced by LL-37 overexpression. Further analysis demonstrated that hCAP18/LL-37 stimulated the phosphorylation of EGFR/HER2 and activated the PI3K/Akt pathway in HCC cells. Furthermore, stronger EGFR/HER2/Akt signals were observed in the PLC/PRF-5xenograft tumor. Interestingly, even though the expression of hCAP18/LL-37 was significantly downregulated in HCC cells and tumors, 1,25(OH)Dtreatment significantly upregulated the hCAP18/LL-37 level both in HCC cells and xenograft tumors. Moreover, 1,25(OH)Dtogether with si-LL-37 significantly enhanced the antitumor activity of 1,25(OH)Din the PLC/PRF-5 xenograft tumor. Collectively, these data suggest that hCAP18/LL-37 promotes HCC cells proliferation through stimulation of the EGFR/HER2/Akt signals and appears to suppress the antitumor activity of 1,25(OH)Din HCC xenograft tumor. This implies that hCAP18/LL-37 may be an important target when aiming to improve the antitumor activity of 1,25(OH)Dsupplementation therapy in HCC.